Aav-based treatment of cholesterol-related disorders

ABSTRACT

The invention in some aspects relates to methods and compositions for assessing the effectiveness of miRNA inhibitors. In other aspects of the invention, methods and compositions for treating cholesterol related disorders are provided. In one aspect of the invention, miRNA inhibitors against miR-122 and rAAV-based compositions comprising the same are provided.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119 of U.S.provisional application U.S. Ser. No. 61/327,383, filed Apr. 23, 2010,and entitled “AAV-based treatment of cholesterol-related disorders,” theentire contents of which are incorporated herein by reference.

GOVERNMENT SUPPORT

This work was supported, at least in part, by grants P01 HL59407-11,2R37GM62862 and 2R01GM65236 from the National Institutes of Health. TheU.S. government has certain rights in this invention.

FIELD OF THE INVENTION

The invention in some aspects relates to methods and compositions forassessing the effectiveness of miRNA inhibitors. In other aspects of theinvention, methods and compositions for treating cholesterol relateddisorders are provided.

BACKGROUND OF INVENTION

Dyslipidemia is associated with defects in cholesterol metabolism andrepresents a major risk factor for cardiovascular disease, the mostcommon cause of morbidity and mortality in the US. One common inheritedform of dyslipidemia is the metabolic defect in low density lipoproteins(LDL) [familial hypercholesterolemia (FH)] caused by genetic mutationsin the LDL receptor (LDLR) gene. MicroRNAs (miRNAs) are small regulatoryRNAs that are important in development and progression of disease. It isunderstood that certain microRNAs have a role cholesterol metabolism. Ahighly abundant miRNA in the liver, miR-122, which does not directlytarget LDLR mRNA, regulates cholesterol metabolism by an unknownmechanism. A locked nucleic acid based oligonucleotide inhibitor ofmiR-122 has been shown to reduce total plasma cholesterol levels in adose dependent manner (See, e.g., Elmen J, et al. Nature, 2008, 452:896-900.) However, such oligonucleotide based inhibitors require dosesimpractical for a therapeutic agent. Furthermore, since theoligonucleotides are administered in finite quantities, repeated

maintain a long term inhibitory effects, which are necessary for manycholesterol-related disorders, like FH. Notwithstanding the link betweenmiRNAs and cholesterol, and prospects of effective therapeutic agentsthat treat cholesterol-related disorders by modulating miRNA function,the development of effective and safe approaches for miRNA inhibition inthe treatment of cholesterol related disorders has been a significantscientific and therapeutic challenge (See, e.g., Czech, MP. N Engl. J.Med. 354; 11 pg. 1144-1145. (2006).)

SUMMARY OF INVENTION

Aspects of the invention are based on molecular sensing systems thatenable the assessment and characterization of miRNA inhibitor functionand thereby facilitate the discovery of miRNA inhibitors that are usefulfor treating and studying disease, e.g., cholesterol-related disorders.According to some aspects of the invention, miRNA inhibitors areidentified herein that are useful for treating cholesterol-relateddisorders. In some embodiments, rAAV-based miRNA inhibitor compositionsare used to effect sustained, tissue specific miRNA inhibition in asubject. In some aspects, a rAAV of the invention harbors at least onetransgene that expresses a miRNA inhibitor that inhibits the function,processing and/or expression of miR-122 in the subject. An exemplarymiRNA inhibitor of the invention has a sequence as set forth in SEQ IDNO: 1.

According to some aspects of the invention, methods are provided fortreating a high cholesterol-related disorder in a subject. In someembodiments, the methods involve administering to a subject an effectiveamount of a rAAV that harbors at least one transgene that expresses amiRNA inhibitor that inhibits the expression of miR-122 in the subject.In some embodiments, the miRNA inhibitor comprises an miR-122 bindingsite. In some embodiments, the miR-122 binding site is flanked by twostem sequences. In some embodiments, the miR-122 binding site comprisesa non-binding, central portion that is not complementary with miR-122,flanked by two portions that are complementary with miR-122. In someembodiments, the miRNA inhibitor comprises a first miR-122 binding siteand a second miR-122 binding site, each binding site flanked by two stemsequences, wherein a first stem sequence flanks the first miR-122binding site at its 5′-end, a second stem sequence flanks the firstmiR-122 binding site at its 3′-end and the second miR-122 binding siteat its 5′-end, and a third stem sequence flanks the second miR-122binding site at its 3′-end. In some embodiments, each of the two miR-122inhibitor binding sites comprises a non-binding, central portion that isnot complementary with miR-122. In some embodiments, the non-binding,central portion of the first miR-122 binding site is at least partiallycomplementary with the non-binding, central portion of the secondmiR-122 binding site. In some embodiments, the non-binding, centralportion of the first miR-122 binding site is complementary with thenon-binding, central portion of the second miR-122 binding site at 1 to5 nucleotides. In some embodiments, the non-binding, central portion ofthe first miR-122 binding site is complementary with the non-binding,central portion of the second miR-122 binding site at 3 nucleotides. Insome embodiments, the non-binding, central portion of the first miR-122binding site has a length in a range of 1 to 10 nucleotides. In someembodiments, the non-binding, central portion of the first miR-122binding site has a length in a range of 3 to 5 nucleotides. In someembodiments, the non-binding, central portion of the first miR-122binding site has a length in a range of 4 nucleotides. In someembodiments, the non-binding, central portion of the second miR-122binding site has a length in a range of 1 to 10 nucleotides. In someembodiments, the non-binding, central portion of the second miR-122binding site has a length in a range of 3 to 5 nucleotides. In someembodiments, the non-binding, central portion of the second miR-122binding site has a length in a range of 4 nucleotides. In someembodiments, the first miR-122 binding and the second miR-122 bindingsite are complementary at a sequence of 2 to 10 nucleotides in length.In some embodiments, the first miR-122 binding and the second miR-122binding site are complementary at a sequence of 4 nucleotides in length.In some embodiments, the miRNA inhibitor comprises two or more miR-122binding sites. In certain embodiments, the miRNA inhibitor has asequence as set forth in SEQ ID NO: 1.

In certain embodiments, the rAAV has a capsid of the AAV9 serotype,which has a sequence as set forth in SEQ ID NO: 3. In some embodiments,the rAAV has a capsid that is a variant of the capsid of the AAV9serotype. In certain embodiments, the rAAV has a capsid of the AAV9serotype variant, Csp-3, which has a sequence as set forth in SEQ ID NO:4. In some embodiments, the rAAV targets liver tissue. In someembodiments, the rAAV transduces hepatocytes. In certain embodiments,the effective amount of rAAV is 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴ genomecopies per kg. In certain embodiments, the effective amount of rAAV is10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ genome copies per subject.

In some embodiments, administering is performed intravenously. In someembodiments, administering is performed by injection into the hepaticportal vein. In some embodiments, the subject is a mouse, a rat, arabbit, a dog, a cat, a sheep, a pig, or a non-human primate. In someembodiments, the subject is a human. In some embodiments, the subject isan animal model of a high cholesterol-related disorder. In someembodiments, the high cholesterol-related disorder is Type I, Type IIa,Type IIb, Type III, Type IV, or Type V Hyperlipoproteinemia. In someembodiments, the high cholesterol-related disorder is associated withdiabetes mellitus, metabolic syndrome, kidney disease (nephroticsyndrome), hypothyroidism, Cushing's syndrome, anorexia nervosa, sleepdeprivation, Zieve's syndrome, antiretroviral drugs, diet, high bodyweight, or low physical activity. In some embodiments, the subject is ahuman and the high cholesterol-related disorder is characterized bytotal serum cholesterol level greater than or equal to 200 mg/dl. Insome embodiments, the subject is a mouse and the highcholesterol-related disorder is characterized by total serum cholesterollevel greater than or equal to 100 mg/dl. In some embodiments, thesubject is a rat and the high cholesterol-related disorder ischaracterized by total serum cholesterol level greater than or equal to70 mg/dl.

According to some aspects of the invention a nucleic acid vector isprovided for assessing the function of a miRNA inhibitor. In someembodiments, the nucleic acid vectors comprise: (a) a first promoteroperably linked with a transgene that comprises: (i.) a protein codingregion, and (ii.) at least one binding site of a test miRNA; and (b) asecond promoter operably linked with a miRNA inhibitor coding region,wherein the miRNA inhibitor hybridizes with the test miRNA. In someembodiments, the first promoter is a RNA Polymerase II promoter. In someembodiments, the second promoter is a RNA Polymerase III promoter. Insome embodiments, the nucleic acid vector further comprises a firstuntranslated region between the first promoter and at least a portion ofthe protein coding region, wherein the second promoter and the miRNAinhibitor coding region are positioned within the first untranslatedregion. In some embodiments, the first untranslated region is positionedat the 5′ end of the complete protein coding region. In someembodiments, the first untranslated region is positioned within anintron of the protein coding region. In some embodiments, the transgenefurther comprises a second untranslated region, wherein the at least onebinding site of the test miRNA is in the second untranslated region. Insome embodiments, the second untranslated region is positioned at the 3′end of the complete protein coding region. In some embodiments, thenucleic acid vector further comprises a pair of inverted terminalrepeats that flank the first promoter and the transgene. In someembodiments, the pair of inverted terminal repeats further flank thesecond promoter and the miRNA inhibitor coding region. In someembodiments, the protein coding region encodes a reporter proteinselected from: a fluorescent protein, luciferase, β-galactosidase,secreted alkaline phosphatase, β-glucuronidase, chloramphenicolacetyltransferase (CAT), and β-lactamase.

In some aspects of the invention, a molecule sensing system is provided.In some embodiments, the molecular sensing system comprises a nucleicacid vector for assessing the function of a miRNA inhibitor. In someembodiments, the nucleic acid vector of the molecular sensing systemcomprises a promoter operably linked with a transgene that is regulatedby a test miRNA and a promoter operably linked with a miRNA inhibitorcoding region.

According to some aspects of the invention, methods are provided forassessing the effectiveness of a miRNA inhibitor. In some embodiments,the methods comprise (a) transfecting a cell with any of the foregoingnucleic acid vectors, wherein the miRNA inhibitor coding region encodesthe miRNA inhibitor; and (b) determining the level of expression of theprotein encoded by the protein coding region in the cell, wherein thelevel of expression of the protein is indicative of the effectiveness ofthe miRNA inhibitor. In some embodiments, the methods further comprisecontacting the cell with the test miRNA. In some embodiments, the cellexpresses the test miRNA. In some embodiments, the methods comprise (a)transfecting a first cell with any one of the foregoing nucleic acidvectors, wherein the miRNA inhibitor coding region encodes the miRNAinhibitor; (b) transfecting a second cell with the nucleic acid vector,wherein levels of the test miRNA are lower in the second cell comparedwith the first cell; and (c) comparing the level of expression of theprotein encoded by the protein coding region in the first cell with thelevel of expression of the protein encoded by the protein coding regionin the second cell, wherein the results of the comparison in (c) areindicative of the effectiveness of the miRNA inhibitor. In someembodiments, the methods comprise (a) transfecting a cell with any oneof the foregoing nucleic acid vectors, wherein the miRNA inhibitorcoding region encodes the miRNA inhibitor; (b) determining a first levelof expression of the protein encoded by the protein coding region in thecell; (c) contacting the cell with the test miRNA; (d) determining asecond level of expression of the protein encoded by the protein codingregion in the cell; and (e) comparing the first level of expression ofthe protein with the second level of expression, wherein the results ofthe comparison in (e) are indicative of the effectiveness of the miRNAinhibitor. In some embodiments, the methods comprise (a) transfecting acell with any one of the foregoing nucleic acid vectors, wherein themiRNA inhibitor coding region encodes the miRNA inhibitor; (b)determining a first level of expression of the protein encoded by theprotein coding region in the cell; and (c) comparing the first level ofexpression of the protein with a control level of expression, whereinthe results of the comparison in (c) are indicative of the effectivenessof the miRNA inhibitor.

In some aspects of the invention, kits are provided for assessing thefunction of a miRNA inhibitor. In some embodiments, the kits comprise acontainer housing any of the foregoing nucleic acid vectors. In someembodiments, the kits comprise a container housing a component of amolecular sensing system.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts a molecular sensing system for evaluating miRNA inhibitorfunction.

FIG. 2A depicts results from a molecular sensing system assay showingthat TuD miR-122 Inhibitor expressed from a polymerase III promoter ishighly effective at derepressing reporter gene expression in 293 cellscompared with other putative miRNA inhibitors.

FIG. 2B depicts results from a molecular sensing system assay showingthat TuD miR-122 Inhibitor expressed from a polymerase III promotercompletely restored reporter gene expression in Huh-7 cells from anucleic acid vector having a single miR122 and substantially derepressedreporter gene expression in Huh-7 cells from a nucleic acid vectorhaving three miR122 binding sites compared with other putative miRNAinhibitors.

FIG. 2C depicts results from an in vivo assay showing that rAAV vectorexpressing a TuD miR-122 Inhibitor effectively knocks down mature freemiR-122 in the liver of mice infected with a rAAV9 containing thevector.

FIG. 3A depicts the sequence and structural features of the TuD miR-122Inhibitor (SEQ ID NO: 1).

FIG. 3B depicts the predicted secondary structure of the TuD miR-122Inhibitor (SEQ ID NO: 1). Structure predicted using MFOLD(mobyle.pasteur.fr/cgi-bin/portal.py?form=mfold)

FIG. 3C depicts the sequence and structural features of the TuD Let-7Inhibitor (SEQ ID NO: 2).

FIG. 4A depicts results from an in vivo assay showing that rAAV vectorexpressing a TuD miR-122 Inhibitor effectively reduced total serumcholesterol levels for up to 10 weeks in mice infected with a rAAV9containing the vector and fed a normal chow diet.

FIG. 4B depicts results from an in vivo assay showing that rAAV vectorexpressing a TuD miR-122 Inhibitor effectively reduced serum HDL levelsfor up to 10 weeks in mice infected with a rAAV9 containing the vectorand fed a normal chow diet.

FIG. 4C depicts results from an in vivo assay showing that rAAV vectorexpressing a TuD miR-122 Inhibitor effectively reduced serum LDL levelsfor up to 2 weeks in mice infected with a rAAV9 containing the vectorand fed a normal chow diet.

FIG. 5A depicts results from an in vivo assay showing that rAAV vectorexpressing a TuD miR-122 Inhibitor effectively reduced total serumcholesterol levels for up to 10 weeks in LDLR^(-/-) Apobec1^(-/-) mice(a model Familial hypercholesterolemia) infected with a rAAV9 containingthe vector.

FIG. 5B depicts results from an in vivo assay showing that rAAV vectorexpressing a TuD miR-122 Inhibitor effectively reduced serum HDL levelsfor up to 2 weeks in LDLR^(-/-) Apobec1^(-/-) mice infected with a rAAV9containing the vector.

FIG. 5C depicts results from an in vivo assay showing that rAAV vectorexpressing a TuD miR-122 Inhibitor effectively reduced serum LDL levelsfor up to 2 weeks in LDLR^(-/-) Apobec1^(-/-) mice for infected with arAAV9 containing the vector.

FIG. 6 Structure of a Tough Decoy miR-122 (TuD) RNA (SEQ ID NO: 5). TuDRNAs contain two single-stranded miRNA binding sites flanked bydouble-stranded stems intended to enhance stability and promote nuclearexport.

FIG. 7 Comparison of miR-122 inhibitor strategies in cultured cells. (a)miRNA inhibitor constructs. (b) Pairing of antagonists to miR-122. Uppersequence in all three panels: miR122 fragment (SEQ ID NO: 6). Sponge:SEQ ID NO: 7. TuD: SEQ ID NO: 8. ZIP: SEQ ID NO: 9. (c) Plasmidharboring nLacZ reporter gene with one or three sites complementary tomiR-122 was co-transfected with pTBG Fluc and either control plasmid,anti-miR-122 sponge plasmid or U6-driven anti-miR-122 TuD plasmid. Thecells were stained for LacZ expression 48 h after transfection, and bluecells were counted. Data are reported relative to a control reporterplasmid lacking miR-122-binding sites. (d) Reporter plasmid expressingnLacZ mRNA containing 3 miR-122-binding sites was co-transfected intoHuH-7 cells with a U6-driven sponge-, miRZip- or TuD-expressing plasmid.The empty plasmid served as the control. (e) HEK 293 cells weretransfected with a nLacZ reporter plasmid containing three fullycomplementary miR-122-binding sites, together with the constructsexpressing anti-let-7 or anti-miR-122 TuD transcribed from a U6 promoteror anti-miR122 sponge or anti-let-7 sponge transcribed from an SV40promoter, as well as different amounts of a plasmid producingpri-miR-122 RNA. Forty-eight hours later, the cells the percentages ofnLacZ positive cells, relative to the control (nLacZ without miR-122binding sites), were determined (c, d, and e). (f) HuH-7 cells weretransfected with reporter plasmid expressing control luciferase,luciferase bearing seven miR-122 binding sites, or seven mutant sites,as well as control plasmid or plasmid expressing anti-miR-122-,anti-let-7 or scrambled TuD RNA. Twenty-four hours later, crude celllysates were prepared and luciferase activity assayed. The data arepresented as mean±standard deviation for firefly luciferase activitynormalized to Renilla luciferase activity.

FIG. 8 Evaluation of let-7 antagonist constructs in HeLa cells. (a,b)Total RNA and protein were prepared from HeLa cells transfected with theconstructs expressing either anti-miR-122 or anti-let-7 TuD, anti-let-7sponge or control plasmid. The relative levels of Dicer mRNA wasmeasured by qRT-PCR (a) and of Dicer protein by Western blotting (b).The figure reports mean±standard deviation.

FIG. 9 Western blot analysis of HeLa cells transfected with theconstructs expressing either anti-miR-122 or anti-let-7 TuD, anti-let-7sponge or plasmid control. Three biological replicates are shown; FIG. 2c reports the quantification of these data.

FIG. 10 Real-time monitoring of endogenous miRNA activity using miRNAsensor system. (a) Schematic presentation of Gaussia luciferase-(Gluc)expressing vectors. CB, chicken β actin promoter with CMV enhancer. AAVvector plasmids were transfected into HuH-7 (b) or HeLa cells (c).Forty-eight hours later, Gluc activity was measured. (d, e) C57BL/6 micewere administered 1×10¹² genome copies of scAAV9 per animal by tail veininjection. Blood was collected at the indicated times and assayed forGluc activity. Gluc expression is reported as mean±standard deviation,relative to samples from mice injected with a scAAV9 vector expressingGluc but lacking both the TuD expression cassette and the 3′ UTRmiRNA-binding sites. Each group had four mice.

FIG. 11 Analysis of miRNA expression in liver from mice administeredscAAV9 expressing anti-miRNA TuD. C57BL/6 mice were injected via tailvein with 1×10¹² genome copies of control, anti-miR-122 or anti-let-7TuD expressing vectors. The animals were sacrificed four weeks later,and total liver RNA was prepared for qRT-PCR (a) and Northern blot (b)analyses of let-7, miR-122, miR-26a, miR-22 and U6. Data are presentedas mean±standard deviation. U6 RNA provided a loading control. (c, d)High throughput sequencing of total liver small RNA was used todetermine the length distribution and abundance of genome-matchingmiR-122 (c) or prefix-matching miR-122 (d) four weeks after scAAVinjection. The most abundant non-genome matching nucleotides added tothe 3′ end of miR-122 fragments are indicated in the grey boxes. (e)Eight let-7 isoforms are expressed in mouse liver. Nucleotidedifferences among the let-7 isoforms are indicated in black and theirpairing to anti-let-7 TuD RNA is shown. The “seed” sequence, animportant feature for miRNA-directed target RNA recognition, isunderlined. Let-7a: SEQ ID NO: 10, Let-7b: SEQ ID NO: 11, Let-7c: SEQ IDNO: 12, Let-7d: SEQ ID NO: 13, Let-7e: SEQ ID NO: 14, Let-7f: SEQ ID NO:15, Let-7g: SEQ ID NO: 16, Let-7i: SEQ ID NO: 17, TuD: SEQ ID NO: 18.(f) The anti-let-7 TuD decreased the abundance of full-length let-7 andincreased the number of prefix-matching let-7 sequence reads, relativeto the control. Isoforms that decreased more than four-fold ingenome-matching reads and increase in prefix-matching reads are shown inblack.

FIG. 12 Northern blot analysis of let-7, miR-122, miR-26a, miR-22 and U6small nucleolar RNA (U6 snoRNA) in total RNA from liver of C57BL/6 miceinjected with 1×10¹² genome copies of scAAV9CBGluc (mock),scAAV9CBGlucTuDmiR-122 (anti-miR-122 TuD) or scAAV9CBGlucTuDlet-7(anti-let-7 TuD) via tail vein injection. The animals were sacrificed 4weeks after injection and total liver RNA was prepared. Three biologicalreplicates are shown and analyzed to generate FIG. 4 b.

FIG. 13 Length distribution and abundance of genome-matching orprefix-matching let-7 isoform sequence reads in liver of mice 4 weeksafter injection of scAAV9CBGluc (mock), scAAV9CBGlucTuD let-7(anti-let-7 TuD). The most abundant non-templated nucleotides added tothe 3′ end of the miR-122 prefixes are indicated in the grey boxes.Let-7a: SEQ ID NO: 10, Let-7b: SEQ ID NO: 11, Let-7c: SEQ ID NO: 12,Let-7d: SEQ ID NO: 13, Let-7e: SEQ ID NO: 14, Let-7f: SEQ ID NO: 15,Let-7g: SEQ ID NO: 16, Let-7i: SEQ ID NO: 17, TuD: SEQ ID NO: 18.

FIG. 14 Abundance of miRNAs in liver of mice 4 weeks after injection ofscAAV9CBGluc (mock), scAAV9CBGlucTuD let-7 (anti-let-7 TuD) orscAAV9CBGlucTuDmiR-122 (anti-miR-122 TuD). Pearson correlation analysiswas performed using GraphPad Prism V5.0b (GraphPad Software, Inc.). Thecorrelation coefficient (r) and p-value are indicated. miRNAs targetedby TuDs are red.

FIG. 15 Expression of natural targets of miR-122 and let-7 inTuD-treated mice. C57BL/6 mice were administered with 1×10¹² genomecopies of control, anti-miR-122 TuD or anti-let-7 TuD scAAV9 vector viatail vein injection. The animals were sacrificed four weeks later andtotal liver (left panel) or heart (right panel) RNA analyzed by qRT-PCRfor representative endogenous targets of miR-122 (Aldolase A, Cyclin G1,Tmed3, Hƒe2, and Cat-1 mRNA) and let-7 (Kras, Hras, Nras, and DicermRNA). The data are presented as the mean percentage (± standarddeviation) of the expression in the mice treated with the control scAAVvector.

FIG. 16 Change in cholesterol profiles of wild-type C57BL/6 andhypercholesterolemic mice (LDLR^(-/-), Apobec1^(-/-)) after miR-122antagonist treatment, relative to control mice. (a) Four-to-six week oldmale wild-type C57BL/6 mice were intravenously injected with 1×10¹²genome copies of scAAV9 per mouse. Serum levels of total cholesterol,high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in thetreated C57B/6 were measured at different time points after injection.(b) The serum transaminases aspartate, aminotransferase (ASL) andalanine aminotransferase (ALT) were assayed to assess liver toxicity.(c) Adult male (n=5 for scrambled and n=6 for anti-miR-122 TuD) andfemale (n=9 for scrambled and n=8 for anti-miR-122 TuD) LDLR^(-/-),Apobec1^(-/-) mice were administered 3×10¹¹ genome copies of scAAV9expressing antimiR-122 by tail vein injection. The changes in totalcholesterol, HDL, and LDL, relative to the control, were measured onemonth later. The figure reports mean±standard deviation.

FIG. 17 Body weights of the study animals. The C57BL/6 wild-type micetreated with 1×10¹²genome copies of scAAV9CBGluc (mock),scAAV9CBGlucTuDmiR-122 (anti-miR-122 TuD) or scAAV9CBGlucTuDlet-7(anti-let-7 TuD) via tail vein injection were weighed 10, 12, 14, 16 and18 weeks later. The data are mean±standard deviation.

DETAILED DESCRIPTION

Aspects of the invention are based on the discovery of miRNA inhibitorsthat are useful for treating and studying cholesterol-related disorders.In some aspects of the invention, a nucleic acid encoding a microRNAinhibitor is packaged in a recombinant AAV (rAAV) for gene transfer to asubject. Recombinant AAVs comprising miRNA inhibitor genes of theinvention are useful for therapeutic purposes as well as for researchpurposes. According to some aspects of the invention, methods areprovided for treating a cholesterol-related disorder in a subject. Insome embodiments, methods of the invention involve administering aneffective amount of a rAAV to a subject. A rAAV may harbor at least onetransgene that expresses a miRNA inhibitor that inhibits the expressionof miR-122 in the subject. An exemplary miRNA inhibitor has a sequenceas set forth in SEQ ID NO: 1.

Cholesterol-Related Disorders

As used herein, a “cholesterol-related disorder” is a condition ordisease that results in a pathological change in cholesterol levels(e.g., pathologically low or pathologically high) in a subject. Asubject may be a mouse, a rat, a rabbit, a dog, a cat, a sheep, a pig,or a non-human primate, for example. A subject may be a human, e.g., asubject having a cholesterol related disorder. In some embodiments, thesubject is an animal model of a high cholesterol-related disorder. Acholesterol-related disorder may be associated with changes in levels oftotal serum cholesterol, serum HDL cholesterol, or serum LDLcholesterol. A cholesterol related disorder may also be associated withalterations in the ratio between serum LDL and HDL (e.g., an LDL/HDLratio). Examples of normal cholesterol ranges for different species areprovided below in Table 1. As is evident from Table 1, normal ranges arespecies dependent. Cholesterol-related disorder associated withabnormally high levels of cholesterol are referred to herein as “highcholesterol-related disorders.” For human subjects a highcholesterol-related disorder may be characterized by total serumcholesterol level greater than 200 mg/dl. For mouse subjects a highcholesterol-related disorder may be characterized by total serumcholesterol level greater than 100 mg/dl. For rat subjects a highcholesterol-related disorder may be characterized by total serumcholesterol level greater than or equal to 70 mg/dl. Other cholesterollevels that are abnormal will be apparent to the skilled artisan.

TABLE 1 Exemplary ranges of normal cholesterol levels. Human Rat MouseTotal cholesterol (mg/dL) 140~199 50~70 ~100 LDL (mg/dL) 105~120  7~115~20 HDL (mg/dL) 30~59 29~40 50~100

Examples of cholesterol-related disorders that may be treated accordingto aspects of the invention include, but are not limited to, Type I,Type II(a and b), Type III, Type IV, and Type V Hyperlipoproteinemia.Further disorders that may be treated according to aspects of theinvention include cholesterol-related disorders associated with diabetesmellitus, metabolic syndrome, kidney disease (nephrotic syndrome),hypothyroidism, Cushing's syndrome, anorexia nervosa, sleep deprivation,Zieve's syndrome, antiretroviral drugs, diet, high body weight, or lowphysical activity. Other cholesterol-related disorders will be apparentto the skilled artisan.

Certain cholesterol-related disorders that may be treated according toaspects of the invention are disorders of a genetic origin (e.g.,inherited, arising from somatic mutations). Familialhypercholesterolemia (FH) (Type II Hyperlipoproteinemia), for example,is a cholesterol-related disorders of genetic origin characterized byhigh cholesterol levels, specifically very high low-density lipoprotein(LDL) levels, in the blood and early cardiovascular disease. Manysubjects with FH have mutations in the LDLR gene that encodes the LDLreceptor protein, which normally removes LDL from the circulation, orapolipoprotein B (ApoB), which is the part of LDL that binds with thereceptor; mutations in other genes are rare. Subjects who have oneabnormal copy (are heterozygous) of the LDLR gene may have prematurecardiovascular disease at the age of 30 to 40.

MiRNA Inhibitors

Micro RNAs (miRNAs) appear to play a role in regulating a broad range ofcellular processes, and changes in miRNA expression have been implicatedin human disease. It is understood that microRNAs have a role in thedevelopment and progression of certain cholesterol-related disorders.The most abundant miRNA in the liver, miR-122 regulates cholesterolmetabolism by an unknown mechanism and does not directly target LDLRmRNA. Although miR-122 represents a potential therapeutic target forhigh cholesterol-related disorders, the prospect of therapeuticallyeffective inhibitors of miR-122 has been largely unfulfilled.

As used herein, the term “miRNA Inhibitor” refers to an agent thatblocks miRNA expression, processing and/or function. A variety of miRNAInhibitor have been disclosed in the art. Non-limiting examples of miRNAinhibitors include but are not limited to microRNA specific antisense,microRNA sponges, tough decoy RNAs (TuD RNAs) and microRNAoligonucleotides (double-stranded, hairpin, short oligonucleotides) thatinhibit miRNA interaction with a Drosha complex. (See, e.g., Ebert, M.S. Nature Methods, Epub Aug. 12, 2007; Takeshi Haraguchi, et al.,Nucleic Acids Research, 2009, Vol. 37, No. 6 e43, the contents of whichrelating to TuD RNAs are incorporated herein by reference).

Molecular Sensing System for miRNA Inhibitors

A molecular sensing system was designed to quantitatively evaluate theinhibitory function of different miRNA inhibitor designs and to enablediscovery of miRNA inhibitors having superior properties compared withmiRNA inhibitors of the art (See FIG. 1 for a schematic of a molecularsensing system). Various miRNA inhibitors were developed and testedusing this system (See, e.g., Example 1). According to some aspects ofthe invention a miRNA inhibitor of miR-122 is identified thateffectively reduces serum cholesterol levels.

A molecular sensing system of the invention typically includescomponents for expressing RNA transcripts of a reporter gene (e.g., aprotein coding gene, e.g., EGFP, Luciferase), the expression of which issensitive to a miRNA that binds to the RNA transcript. Typically, theRNA transcript is an mRNA transcript encoding a protein. Thus, reportergene activity is often assessed by detecting levels of a protein encodedby an mRNA transcript of the transgene. However, the RNA transcript ofthe reporter gene may itself serve as a reporter of transgene activity.For example, the RNA may be detected using any one of a variety ofstandard RNA detection strategies, e.g. RT-PCR, and thus, may serve as areporter for activity of the transgene. Typically, the RNA transcript ofthe transgene bears one or more miRNA binding sites. Thus, whenexpressed in a cell, RNA transcripts of a molecular sensing system aretypically sensitive to the presence of miRNA molecules of the cell thatbind to them at miRNA binding sites. The miRNA binding sites aretypically in the 3′ end of the transcript. However, a miRNA binding sitemay be in a coding region or in any untranslated region of the transgeneprovided that when a miRNA binds to the site in a cell having afunctional miRNA gene silencing pathway, or a in vitro system thatrecapitulates miRNA activity, expression of the transcript is inhibited.The molecule sensing system also typically comprises components forexpressing a test miRNA inhibitor. When mRNA transcripts bearing bindingsites for a miRNA are expressed in the presence of the miRNA, the miRNAhybridizes to the binding sites and inhibits expression of a reporterprotein encoded by the mRNA. However, when a miRNA inhibitor isexpressed that blocks function of the miRNA, expression of the reporterprotein is not inhibited (or inhibition of expression is attenuated).Thus, molecular sensing systems of the invention enable efficientscreening and identification of miRNA inhibitors with effectiveinhibitory properties based on levels of reporter gene expression.

A molecule sensing system often includes a nucleic acid vectorcomprising a promoter operably linked with a transgene that is regulatedby a miRNA and a promoter operably linked with an miRNA inhibitor codingregion. The transgene of the nucleic acid vector typically includes, ata minimum, a protein coding region (e.g., a reporter protein codingregion) and at least one binding site of a miRNA. The protein codingregion may encode a reporter protein such as, for example, a fluorescentprotein, (e.g., GFP, dsRed, etc.) luciferase, β-galactosidase, secretedalkaline phosphatase, β-glucuronidase, chloramphenicol acetyltransferase(CAT), and β-lactamase. The promoter for the transgene and the promoterfor the miRNA inhibitor coding region may be the same promoter or may bedifferent promoters. The promoter for the transgene is typically a RNAPolymerase II promoter. The promoter for the miRNA inhibitor may be aRNA Polymerase II promoter or an RNA Polymerase III promoter (e.g., a U6promoter).

The skilled artisan will appreciate that the promoter operably linkedwith the transgene may be positioned anywhere within the nucleic acidvector provided that the transgene is capable of being expressed in anappropriate expression system, e.g., in a cell or an in vitrotranscription/translation system. Similarly, the skilled artisan willappreciate that the promoter operably linked with the miRNA inhibitorcoding region may be positioned anywhere within the nucleic acid vectorprovided that the miRNA inhibitor coding region is capable of beingexpressed in an appropriate expression system, e.g., in a cell or an invitro transcription/translation system. For example, the second promoteroperably linked with a miRNA inhibitor coding region may be positionedupstream of the first promoter operably linked with the transgene(5-prime relative to the first promoter operably linked with thetransgene.) The second promoter operably linked with a miRNA inhibitorcoding region may be positioned downstream of the first promoteroperably linked with the transgene (3-prime relative to the firstpromoter operably linked with the transgene.) The second promoteroperably linked with a miRNA inhibitor coding region may be positionedbetween the first promoter and the transgene coding region (e.g., withinan intron). The second promoter operably linked with a miRNA inhibitorcoding region may be positioned within any intron of the transgene. Thesecond promoter operably linked with a miRNA inhibitor coding region maybe positioned within a untranslated region upstream of the transgenecoding region (e.g., a 5′-UTR) or downstream of the transgene codingregion (e.g., a 3′-UTR).

A molecule sensing system may include, for example, a nucleic acidvector comprising a first promoter operably linked with a transgene thatis regulated by a test miRNA and a second promoter operably linked witha miRNA inhibitor coding region. The nucleic acid vector may be arecombinant viral genome. For example, the nucleic acid vector may be arecombinant AAV vector. Accordingly, the nucleic acid vector further mayinclude a pair of inverted terminal repeats that flank the promoteroperably linked with transgene. The pair of inverted terminal repeatsmay further flank the promoter operably linked with the miRNA inhibitorcoding region.

Methods are provided for assessing the effectiveness of a miRNAinhibitor using a molecular sensing system of the invention. The methodstypically involve (a) transfecting a cell with a nucleic acid vector,which comprises a first promoter operably linked with a transgene thatcomprises a protein coding region and at least one binding site of amiRNA and a second promoter operably linked with a coding region for amiRNA inhibitor that hybridizes with the miRNA, and (b) determining thelevel of expression of the protein encoded by the protein coding regionin the cell. The level of expression of the protein is indicative of theeffectiveness of the miRNA inhibitor. For example, when the nucleic acidvector is transfected in a cell that expresses the miRNA, the miRNA willbind to its cognate binding site(s) in the mRNA transcribed from thetransgene and inhibit expression of the mRNA. If the miRNA inhibitor iseffective, it will block (or decrease) the activity of the miRNA, e.g.,by hybridizing with the miRNA, and relieve (or attenuate) repression ofexpression of the mRNA. Changes in expression of the mRNA are typicallyobserved by assessing levels of the reporter protein encoded by themRNA. Thus, different miRNA inhibitors can be compared based on reporterprotein levels. As will be appreciated by the skilled artisan, thesystem can be tuned in various ways to identify inhibitors havingdesired levels of effectiveness. For example, the quality of the miRNAbinding site on the transgene mRNA can be designed or selected. Highquality binding sites, e.g., binding sites that bind to the test miRNAwith high affinity can be designed or selected. Binding sites can bedesigned de novo or selected from miRNA bindings sites of known genes(e.g., an miR-122 binding site on Cyclin G may be selected). The numberof miRNA binding sites in the transgene mRNA can also be altered. Forexample, multiple binding sites can be used or a single binding site canbe used. By adjusting parameters such as the affinity of the miRNA forbinding to its miRNA and the number of bindings sites, it becomespossible to increase or decrease the stringency with which miRNAinhibitors are selected. For example, high quality miRNA inhibitors canbe selected by using a transgene having multiple high-quality bindingssites for a test miRNA. The molecule sensing system may be used in an invitro expression system or in cells.

The level of the miRNA is another example of a parameter that can bemodulated to increase or decrease the stringency with which miRNAinhibitors are selected. Thus, the methods may further comprisecontacting cells with the miRNA or adding miRNA to an in vitroexpression system. Multiple experiments may be performed, e.g., inparallel, using different doses of the miRNA to enable an evaluation ofthe dose dependent inhibition properties of the miRNA inhibitors.

Any of a variety of control values or experiments may be obtained orperformed to assess the effectiveness of a test miRNA inhibitor. Themethods may comprise (a) transfecting a first cell with a nucleic acidvector of a molecular sensing system, wherein the miRNA inhibitor codingregion of the vector encodes the miRNA inhibitor; (b) transfecting asecond cell with the nucleic acid vector, wherein levels of the testmiRNA are lower in the second cell compared with the first cell; and (c)comparing the level of expression of the protein encoded by the proteincoding region in the first cell with the level of expression of theprotein encoded by the protein coding region in the second cell, whereinthe results of the comparison in (c) are indicative of the effectivenessof the miRNA inhibitor. The methods may comprise (a) transfecting a cellwith any one of the foregoing nucleic acid vectors, wherein the miRNAinhibitor coding region encodes the miRNA inhibitor; (b) determining afirst level of expression of the protein encoded by the protein codingregion in the cell; (c) contacting the cell with the test miRNA; (d)determining a second level of expression of the protein encoded by theprotein coding region in the cell; and (e) comparing the first level ofexpression of the protein with the second level of expression, whereinthe results of the comparison in (e) are indicative of the effectivenessof the miRNA inhibitor.

MiRNA Inhibitor Structure

Aspects of the invention are based on the discovery of miRNA inhibitorsthat target miR-122 and block its function. For example, high qualitymiRNA inhibitors have been discovered using a molecular sensing systemof the invention.

The typical miRNA inhibitor of the invention is a nucleic acid moleculethat comprises at least one miRNA binding site, e.g., an miR-122 bindingsite. The miRNA inhibitors may comprise 1 miRNA binding site, 2 miRNAbinding sites, 3 miRNA binding sites, 4 miRNA binding sites, 5 miRNAbinding sites, 6 miRNA binding sites, 7 miRNA binding sites, 8 miRNAbinding sites, 9 miRNA binding sites, 10 miRNA binding sites, or moremiRNA binding sites. As used herein, the term “miRNA binding site,” withreference to a miRNA inhibitor, refers to a sequence of nucleotides in amiRNA inhibitor that are sufficiently complementary with a sequence ofnucleotides in a miRNA to effect base pairing between the miRNAinhibitor and the miRNA. Typically, a miRNA binding site comprises asequence of nucleotides that are sufficiently complementary with asequence of nucleotides in a miRNA to effect base pairing between themiRNA inhibitor and to thereby inhibit binding of the miRNA to a targetmRNA.

As used herein the term “complementary” or “complementarity” refers tothe ability of a nucleic acid to form hydrogen bond(s) with another RNAsequence by either traditional Watson-Crick or other non-traditionalbase pairing. In reference to the nucleic molecules of the presentinvention, the binding free energy for a nucleic acid molecule with itstarget or complementary sequence is sufficient to allow the relevantfunction of the nucleic acid to proceed, e.g., miRNA inhibition.Determination of binding free energies for nucleic acid molecules iswell known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant.Biol. LII pp. 123 133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA83:9373 9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). Apercent complementarity indicates the percentage of contiguous residuesin a nucleic acid molecule which can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” means that all the contiguousresidues of a nucleic acid sequence will hydrogen bond with the samenumber of contiguous residues in a second nucleic acid sequence. In someembodiments the nucleic acids have 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% complementarity.

For a miRNA inhibitor having two miRNA binding sites, the first miRNAbinding site and the second miRNA binding site may be complementary,e.g., at a sequence of 2 to 10 nucleotides in length. In one example,the first miRNA binding and the second miRNA binding site arecomplementary at a sequence of 4 nucleotides in length. Each miRNAbinding site of a miRNA inhibitor may be any of a variety of lengths.For example, the miRNA binding site of a miRNA inhibitor may be 5nucleotides to 35 nucleotides, 10 nucleotides to 30 nucleotides, or 15nucleotides to 25 nucleotides. Typically the length of the miRNA bindingsite depends on the length and/or structure of the miRNA to which itbinds.

Often a miRNA binding site of a miRNA inhibitor of the invention isflanked by one or more stem sequence. As used herein the term “stemsequence” refers to a sequence of a nucleic acid that results inintramolecular base pairing. In some embodiments, stem sequences are notcomplementary with a target miRNA. Intramolecular base pairing may occurwhen two stem sequence regions of a miRNA inhibitor, usually palindromicsequences, base-pair to form a double helix, which may end in anunpaired loop. Thus, based pairing may form within a stem sequence orbetween two stem sequences. A stem sequence may be of a variety oflengths. For example, a stem sequence may be in range 3 nucleotides to200 nucleotides, 3 nucleotides to 100 nucleotides, 3 nucleotides to 50,3 nucleotides to 25 nucleotides, 10 nucleotides to 20 nucleotides, 20nucleotides to 30 nucleotides, 30 nucleotides to 40 nucleotides, 40nucleotides to 50 nucleotides, or 50 nucleotides to 100 nucleotides. Astem sequence may be up to 5 nucleotides, up to 10 nucleotides, up to 20nucleotides, up to 50 nucleotides, up to 100 nucleotides, up to 200nucleotides, or more. Linker sequences may also be included in a miRNAinhibitor. The miRNA inhibitor may comprise a first miRNA binding siteand a second miRNA binding site, each binding site flanked by two stemsequences. A first stem sequence may flank the first miRNA binding siteat its 5′-end, a second stem sequence may flank the first miRNA bindingsite at its 3′-end and the second miRNA binding site at its 5′-end, anda third stem sequence may flank the second miRNA binding site at its3′-end. The skilled artisan will readily envision other configurationsof binding sites and flanking stem sequences.

The miRNA binding site of a miRNA inhibitor of the invention maycomprise a non-binding, central portion that is not complementary withthe target miRNA (e.g., miR-122), flanked by two portions that arecomplementary with the target miRNA. A non-binding, central portion thatis not complementary with the target miRNA need not be perfectlycentered within the miRNA binding site. For example, a non-bindingcentral portion may be flanked on either side by portions that arecomplementary with the target miRNA that are of different lengths. AmiRNA inhibitor of the invention may comprise multiple miRNA bindingsites that have a non-binding, central portion that is not complementarywith the target miRNA. The non-binding, central portion of a miRNAbinding site may have any of a variety of lengths. For example, anon-binding, central portion of a miRNA binding site may be in a rangeof 1 nucleotide to 20 nucleotides, 1 nucleotide to 10 nucleotides, 1nucleotide to 5 nucleotides. The non-binding, central portion of a miRNAbinding site may have a length in a range of 3 to 5 nucleotides. In oneexample, the non-binding, central portion of a miRNA binding site has alength of 4 nucleotides. The length of the non-binding, central portionwill typically depend on the length of the miRNA binding site.

Often the non-binding, central portion of a first miRNA binding site isat least partially complementary with the non-binding, central portionof a second miRNA binding site of the inhibitor. Thus, two binding sitesof an inhibitor may base pair (hybridize) with each other. Thenon-binding, central portion of a first miRNA binding site of aninhibitor may be complementary with the non-binding, central portion ofa second miRNA binding site of an inhibitor at, for example, 2nucleotides to 10 nucleotides, depending on the length of the bindingsite and the non-binding central portion. The non-binding, centralportion of a first miRNA binding site of an inhibitor may becomplementary with the non-binding, central portion of a second miRNAbinding site at, for example, 2 nucleotides, 3 nucleotides, 4nucleotides, 5 nucleotides, 10 nucleotides, or more nucleotides,typically depending on the length of the binding site and thenon-binding central portion.

Some aspects of this invention provide miRNA inhibitors that target aplurality of miRNAs. In some embodiments, targeting a plurality ofmiRNAs circumvents the problem of inhibition of an individual miRNAbeing compensated for by related miRNAs. In some embodiments, theplurality of miRNAs belong to a family of miRNAs, for example, the let-7family. In some embodiments, the plurality of miRNAs share at least somesequence identity. For example, in some embodiments, the plurality ofmiRNAs each comprise at least one stretch of 5 or more nucleotides thatis identical across all of the plurality of miRNAs. In some embodiments,the plurality of miRNAs each comprise at least one stretch of 5 or morenucleotides that is at least 70%, at least 80%, at least 90%, at least95%, or at least 98% identical to the consensus sequence of that stretchof nucleotides of the plurality of target miRNAs.

The term “consensus sequence,” as used herein, refers to a sequence ofnucleotides that reflects the most common nucleotide shared by multiplenucleotide sequences at a specific position. In some embodiments, themultiple nucleotide sequences are related nucleotide sequences, forexample, sequences of members of the same miRNA family. In someembodiments, a consensus sequence is obtained by aligning two or moresequences and determining the nucleotide most commonly found or mostabundant in the aligned sequences at a particular position. Methods andalgorithms for sequence alignment for obtaining consensus sequences froma plurality of sequences are well known to those of skill in the art andthe invention is not limited in this respect.

In some embodiments, the miRNA inhibitor targeting a plurality of miRNAsis TuD comprising at least one miRNA binding site complementary to aconsensus sequence of the plurality of miRNAs. In some embodiments, theconsensus sequence is at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, orat least 20 nucleotides in length. In some embodiments, the miRNAinhibitor comprises a first miRNA binding site and a second miRNAbinding site, wherein a first stem sequence flanks the first miRNAbinding site at its 5′-end, a second stem sequence flanks the firstmiRNA binding site at its 3′-end and the second miRNA binding site atits 5′-end, and a third stem sequence flanks the second miRNA bindingsite at its 3′-end, wherein at least one of the miRNA binding sitescomprises a nucleotide sequence complementary to a consensus sequence ofthe plurality of target miRNAs. In some embodiments, the first and thesecond miRNA binding sites are complementary to a consensus sequence ofthe plurality of target miRNAs. In some embodiments, the first and/orthe second miRNA binding site is at least 7-%, at least 80%, at least90%, at least 95%, or at least 98% complementary to a consensus sequenceof the plurality of target miRNAs. In some embodiments, the consensussequence the first miRNA binding site is complementary to is directlyadjacent to the consensus sequence the second miRNA binding site iscomplementary to.

In some embodiments, a miRNA inhibitor is provided that targets aplurality of let-7 family member miRNAs. In some embodiments, the miRNAinhibitor comprises a sequence of at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, or 26, contiguous nucleotides of SEQ ID NO: 18. In someembodiments, the miRNA inhibitor comprises or consists of the nucleotidesequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO:24. In some embodiments, methods are provided that comprise contacting acell with an miRNA inhibitor. The cell may be in vitro or may be invivo. Accordingly, in some embodiments, the methods involve adding amiRNA inhibitor to a culture of cells in vitro. In other embodiments,the methods involve administering a miRNA inhibitor to a subject.

Some aspects of this invention provide a method of generating a miRNAinhibitor targeting a plurality of miRNAs, wherein the method comprisesobtaining a consensus sequence of the plurality of target miRNAs, andgenerating a miRNA inhibitor, for example, a miRNA inhibitor describedherein (e.g., a TuD), comprising a miRNA binding site able to bind tothe consensus sequence, and, thus, targeting the plurality of miRNAs. Insome embodiments, the miRNA inhibitor so generated comprises a firstmiRNA binding site and a second miRNA binding site, wherein a first stemsequence flanks the first miRNA binding site at its 5′-end, a secondstem sequence flanks the first miRNA binding site at its 3′-end and thesecond miRNA binding site at its 5′-end, and a third stem sequenceflanks the second miRNA binding site at its 3′-end, wherein the miRNAinhibitor comprises a nucleotide sequence complementary to the consensussequence of the plurality of target miRNAs. In some embodiments, themethod further comprises synthesizing the miRNA inhibitor targeting aplurality of miRNAs.

Recombinant AAVs

It has been discovered that the miRNA inhibitors of the invention whenexpressed from a recombinant AAV vector achieve long-term miRNAinhibitory effects in a subject. For example, it has been discoveredthat a miRNA inhibitor against miR-122 delivered using a rAAV to anormal subject (who does not have a cholesterol-related disorder)significantly reduces total serum cholesterol in the subject for asustained period of time, e.g., up to at least 14 weeks. It has furtherbeen discovered that a miRNA inhibitor against miR-122 delivered using arAAV to a subject having a high cholesterol-related disorder alsosignificantly reduces total serum cholesterol in the subject for asustained period of time, e.g., up to at least 14 weeks.

AAVs are natural inhabitants in mammals. AAVs isolated from mammals,particularly non-human primates, are useful for creating gene transfervectors for clinical development and human gene therapy applications. Inaspects of the invention, a recombinant AAV9 achieves efficient andstable miR-122 antagonism in normal C57BL/6 mice by expressing anoptimized miR-122 inhibitor (also referred to herein as an miR-122antagonist (Antag)). A single intravenous injection of a rAAV9comprising a rAAV vector encoding an miR-122 inhibitor(rAAV9-miR-122Antag) produced an significant decrease in the level ofmature miR-122 and significant up-regulation of miR-122 target genes. Areduction in total serum cholesterol, HDL, and LDL of up to about 50%was observed in a normal subject who was fed a regular diet.

In some aspects, the invention provides isolated AAVs. As used hereinwith respect to AAVs, the term “isolated” refers to an AAV that has beenisolated from its natural environment (e.g., from a host cell, tissue,or subject) or artificially produced. Isolated AAVs may be producedusing recombinant methods. Such AAVs are referred to herein as“recombinant AAVs”. Recombinant AAVs (rAAVs) preferably havetissue-specific targeting capabilities, such that a transgene of therAAV will be delivered specifically to one or more predeterminedtissue(s). The AAV capsid is an important element in determining thesetissue-specific targeting capabilities. Thus, a rAAV having a capsidappropriate for the tissue being targeted can be selected. Typically,the rAAV has a capsid that has a tropism for (that targets) livertissue, particularly hepatocytes of liver tissue. For example, the rAAVcapsid may be of the AAV9 serotype, which has a sequence as set forth inSEQ ID NO: 3, or a variant thereof. The rAAV has a capsid of the AAV9serotype variant, Csp-3, which has a sequence as set forth in SEQ ID NO:4. Examples of AAV9 serotype variants are disclosed in U.S. ProvisionalApplication Ser. No. 61/182,084, filed May 28, 2009, the contents ofwhich relating to AAV capsid sequences are incorporated herein byreference. Still, in some embodiments the AAV serotype is selected from:AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAVrh.10. In otherembodiments the AAV serotype is a variant of an AAV serotype is selectedfrom: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAVrh.10.

>gi|46487805|gb|AAS99264.1| capsid protein VP1 [Adeno-associatedvirus 9] (SEQ ID NO: 3)MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL >capsid protein VP1 [Adeno-associated virus]CSp3 (SEQ ID NO: 4)MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTIASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKRISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIRVKEVTDNNGVKTITNNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTRNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGVKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

Recombinant AAVs: Production Methods

Methods for obtaining recombinant AAVs having a desired capsid proteinare well known in the art. (See, for example, US 2003/0138772, thecontents of which are incorporated herein by reference in theirentirety). Typically the methods involve culturing a host cell whichcontains a nucleic acid sequence encoding an AAV capsid protein orfragment thereof; a functional rep gene; a recombinant AAV vectorcomposed of, AAV inverted terminal repeats (ITRs) and a transgene; andsufficient helper functions to permit packaging of the recombinant AAVvector into the AAV capsid proteins.

The components to be cultured in the host cell to package a rAAV vectorin an AAV capsid may be provided to the host cell in trans.Alternatively, any one or more of the required components (e.g.,recombinant AAV vector, rep sequences, cap sequences, and/or helperfunctions) may be provided by a stable host cell which has beenengineered to contain one or more of the required components usingmethods known to those of skill in the art. Most suitably, such a stablehost cell will contain the required component(s) under the control of aninducible promoter. However, the required component(s) may be under thecontrol of a constitutive promoter. Examples of suitable inducible andconstitutive promoters are provided herein, in the discussion ofregulatory elements suitable for use with the transgene. In stillanother alternative, a selected stable host cell may contain selectedcomponent(s) under the control of a constitutive promoter and otherselected component(s) under the control of one or more induciblepromoters. For example, a stable host cell may be generated which isderived from 293 cells (which contain El helper functions under thecontrol of a constitutive promoter), but which contain the rep and/orcap proteins under the control of inducible promoters. Still otherstable host cells may be generated by one of skill in the art.

The recombinant AAV vector, rep sequences, cap sequences, and helperfunctions required for producing the rAAV of the invention may bedelivered to the packaging host cell using any appropriate geneticelement (vector). The selected genetic element may be delivered by anysuitable method, including those described herein. The methods used toconstruct any

are known to those with skill in nucleic acid manipulation and includegenetic engineering, recombinant engineering, and synthetic techniques.See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods ofgenerating rAAV virions are well known and the selection of a suitablemethod is not a limitation on the present invention. See, e.g., K.Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

In some embodiments, recombinant AAVs may be produced using the tripletransfection method (described in detail in U.S. Pat. No. 6,001,650).Typically, the recombinant AAVs are produced by transfecting a host cellwith an recombinant AAV vector (comprising a transgene) to be packagedinto AAV particles, an AAV helper function vector, and an accessoryfunction vector. An AAV helper function vector encodes the “AAV helperfunction” sequences (i.e., rep and cap), which function in trans forproductive AAV replication and encapsidation. Preferably, the AAV helperfunction vector supports efficient AAV vector production withoutgenerating any detectable wild-type AAV virions (i.e., AAV virionscontaining functional rep and cap genes). Non-limiting examples ofvectors suitable for use with the present invention include pHLP19,described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described inU.S. Pat. No. 6,156,303, the entirety of both incorporated by referenceherein. The accessory function vector encodes nucleotide sequences fornon-AAV derived viral and/or cellular functions upon which AAV isdependent for replication (i.e., “accessory functions”). The accessoryfunctions include those functions required for AAV replication,including, without limitation, those moieties involved in activation ofAAV gene transcription, stage specific AAV mRNA splicing, AAV DNAreplication, synthesis of cap expression products, and AAV capsidassembly. Viral-based accessory functions can be derived from any of theknown helper viruses such as adenovirus, herpes virus (other than herpessimplex virus type-1), and vaccinia virus.

In some aspects, the invention provides transfected host cells. The term“transfection” is used to refer to the uptake of foreign DNA by a cell,and a cell has been “transfected” when exogenous DNA has been introducedinside the cell membrane. A number of transfection techniques aregenerally known in the art. See, e.g., Graham et al. (1973) Virology,52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual,Cold Spring Harbor Laboratories, New York, Davis et al. (1986) BasicMethods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousnucleic acids, such as a nucleotide integration vector and other nucleicacid molecules, into suitable host cells. Transfection may be achievefor example by infecting a cell with a rAAV harboring a rAAV vector.

A “host cell” refers to any cell that harbors, or is capable ofharboring, a substance of interest. Often a host cell is a mammaliancell. A host cell may be used as a recipient of an AAV helper construct,an AAV transgene plasmid, e.g., comprising a promoter operably linkedwith a miRNA inhibitor, an accessory function vector, or other transferDNA associated with the production of recombinant AAVs. The termincludes the progeny of the original cell which has been transfected.Thus, a “host cell” as used herein may refer to a cell which has beentransfected with an exogenous DNA sequence. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

As used herein, the term “cell line” refers to a population of cellscapable of continuous or prolonged growth and division in vitro. Often,cell lines are clonal populations derived from a single progenitor cell.It is further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants.

As used herein, the terms “recombinant cell” refers to a cell into whichan exogenous DNA segment, such as DNA segment that leads to thetranscription of a biologically-active polypeptide or production of abiologically active nucleic acid such as an RNA, has been introduced.

As used herein, the term “vector” includes any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, artificial chromosome,virus, virion, etc., which is capable of replication when associatedwith the proper control elements and which can transfer gene sequencesbetween cells. Thus, the term includes cloning and expression vehicles,as well as viral vectors. In some embodiments, useful vectors arecontemplated to be those vectors in which the nucleic acid segment to betranscribed is positioned under the transcriptional control of apromoter. A “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. The phrases“operatively positioned,” “under control” or “under transcriptionalcontrol” means that the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene. The term “expression vector orconstruct” means any type of genetic construct containing a nucleic acidin which part or all of the nucleic acid encoding sequence is capable ofbeing transcribed. In some embodiments, expression includestranscription of the nucleic acid, for example, to generate abiologically-active polypeptide product or inhibitory RNA (e.g., shRNA,miRNA, miRNA inhibitor) from a transcribed gene.

The foregoing methods for packaging recombinant vectors in desired AAVcapsids to produce the rAAVs of the invention are not meant to belimiting and other suitable methods will be apparent to the skilledartisan.

Recombinant AAV Vectors

“Recombinant AAV (rAAV) vectors” of the invention are typically composedof, at a minimum, a transgene, e.g., encoding a miRNA inhibitor or anucleic acid of a molecular sensing system, and its regulatorysequences, and 5′ and 3′ AAV inverted terminal repeats (ITRs). It isthis recombinant AAV vector which is packaged into a capsid protein anddelivered to a selected target cell. In some embodiments, the transgeneis a nucleic acid sequence, heterologous to the vector sequences, whichencodes a miRNA inhibitor. The nucleic acid coding sequence isoperatively linked to regulatory components in a manner which permitstransgene transcription, translation, and/or expression in a cell of atarget tissue. Recombinant AAV based vectors may be developed fortargeting the miRNA inhibitors to liver tissue to interfere with miR-122function and reduced cholesterol levels. Recombinant AAV based vectorsmay also be developed for targeting a nucleic vector of a molecularsensing system to cell for evaluating or screening miRNA inhibitors inthe cell.

The AAV sequences of the vector typically comprise the cis-acting 5′ and3′ inverted terminal repeat sequences (See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168(1990)). The ITR sequences are about 145 by in length. Preferably,substantially the entire sequences encoding the ITRs are used in themolecule, although some degree of minor modification of these sequencesis permissible. The ability to modify these ITR sequences is within theskill of the art. (See, e.g., texts such as Sambrook et al, “MolecularCloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory,New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). Anexample of such a molecule employed in the present invention is a“cis-acting” plasmid containing the transgene, in which the selectedtransgene sequence and associated regulatory elements are flanked by the5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained fromany known AAV, including presently identified mammalian AAV types.

In addition to the major elements identified above for the recombinantAAV vector, the vector also includes conventional control elementsnecessary which are operably linked to the transgene in a manner whichpermits its transcription, translation and/or expression in a celltransfected with the plasmid vector or infected with the virus producedby the invention. As used herein, “operably linked” sequences includeboth expression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

As used herein, a nucleic acid sequence (e.g., coding sequence) andregulatory sequences are said to be “operably” linked when they arecovalently linked in such a way as to place the expression ortranscription of the nucleic acid sequence under the influence orcontrol of the regulatory sequences. If it is desired that the nucleicacid sequences be translated into a functional protein, two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not interfere with the ability of the promoter region to direct thetranscription of the coding sequences or interfere with the function ofthe corresponding RNA transcript. Thus, a promoter region would beoperably linked to a nucleic acid sequence if the promoter region werecapable of effecting transcription of that DNA sequence such that theresulting transcript might become a functional RNA molecule (e.g., aproperly folded miRNA inhibitor).

In some embodiments, the regulatory sequences impart tissue-specificgene expression capabilities. In some cases, the tissue-specificregulatory sequences bind tissue-specific transcription factors thatinduce transcription in a tissue specific manner. Such tissue-specificregulatory sequences (e.g., promoters, enhancers, etc.) are well knownin the art. Often, a miRNA inhibitor is expressed from a polymerase IIIpromoter, such as, for example, a U6 promoter. However, otherappropriate promoters, e.g., RNA polymerase II promoters, may be used.

Recombinant AAV Administration Methods

The rAAVs may be delivered to a subject in compositions according to anyappropriate methods known in the art. The rAAV, preferably suspended ina physiologically compatible carrier (i.e., in a composition), may beadministered to a subject, such as, for example, a human, mouse, rat,cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster,chicken, turkey, or a non-human primate (e.g., Macaque).

The rAAVs are administered in sufficient amounts to transfect the cellsof a desired tissue and to provide sufficient levels of gene transferand expression without undue adverse effects. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the selected organ (e.g., intraportaldelivery to the liver), oral, inhalation (including intranasal andintratracheal delivery), intraocular, intravenous, intramuscular,subcutaneous, intradermal, intratumoral, and other parental routes ofadministration. In certain circumstances it will be desirable to deliverthe rAAV-based therapeutic constructs in suitably formulatedpharmaceutical compositions disclosed herein either subcutaneously,intraopancreatically, intranasally, parenterally, intravenously,intramuscularly, intrathecally, or orally, intraperitoneally, or byinhalation. In some embodiments, the administration modalities asdescribed in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (eachspecifically incorporated herein by reference in its entirety) may beused to deliver rAAVs.

Delivery of the rAAVs to a mammalian subject may be by intravenousinjection. In some embodiments, the mode of administration of rAAVs isby portal vein injection. Administration into the bloodstream may be byinjection into a vein, an artery, or any other vascular conduit. In someembodiments, administration of rAAVs into the bloodstream is by way ofisolated limb perfusion, a technique well known in the surgical arts,the method essentially enabling the artisan to isolate a limb from thesystemic circulation prior to administration of the rAAV virions. Avariant of the isolated limb perfusion technique, described in U.S. Pat.No. 6,177,403, can also be employed by the skilled artisan to administerthe rAAVs into the vasculature of an isolated limb to potentiallyenhance transduction into muscle cells or tissue. Routes ofadministration may be combined, if desired.

Moreover, in certain instances, it may be desirable to deliver thevirions to the CNS of a subject. By “CNS” is meant all cells and tissueof the brain and spinal cord of a vertebrate. Thus, the term includes,but is not limited to, neuronal cells, glial cells, astrocytes,cereobrospinal fluid (CSF), interstitial spaces, bone, cartilage and thelike. Recombinant AAVs may be delivered directly to the CNS or brain byinjection into, e.g., the ventricular region, as well as to the striatum(e.g., the caudate nucleus or putamen of the striatum), spinal cord andneuromuscular junction, or cerebellar lobule, with a needle, catheter orrelated device, using neurosurgical techniques known in the art, such asby stereotactic injection (see, e.g., Stein et al., J Virol73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidsonet al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. GeneTher. 11:2315-2329, 2000).

The compositions of the invention may comprise a rAAV alone, or incombination with one or more other viruses (e.g., a second rAAV encodinghaving one or more different transgenes, e.g., one or more differentmiRNA inhibitors). In some embodiments, a compositions comprise 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more different rAAVs each having one or moredifferent transgenes.

Suitable carriers may be readily selected by one of skill in the art inview of the indication for which the rAAV is directed. For example, onesuitable carrier includes saline, which may be formulated with a varietyof buffering solutions (e.g., phosphate buffered saline). Otherexemplary carriers include sterile saline, lactose, sucrose, calciumphosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, andwater. The selection of the carrier is not a limitation of the presentinvention.

Optionally, the compositions of the invention may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

The dose of rAAV virions required to achieve a particular “therapeuticeffect,” e.g., the units of dose in genome copies/per kilogram of bodyweight (GC/kg), will vary based on several factors including, but notlimited to: the route of rAAV virion administration, the level of geneor RNA expression required to achieve a therapeutic effect, the specificdisease or disorder being treated, and the stability of the gene or RNAproduct. One of skill in the art can readily determine a rAAV viriondose range to treat a subject having a particular disease or disorderbased on the aforementioned factors, as well as other factors that arewell known in the art.

An effective amount of a rAAV is an amount sufficient to target infectan animal, target a desired tissue. In some embodiments, an effectiveamount of a rAAV is an amount sufficient to produce a stable somatictransgenic animal model. The effective amount will depend primarily onfactors such as the species, age, weight, health of the subject, and thetissue to be targeted, and may thus vary among animal and tissue. Forexample, a effective amount of the rAAV is generally in the range offrom about 1 ml to about 100 ml of solution containing from about 10⁹ to10¹⁶ genome copies. In some cases, a dosage between about 10¹¹ to 10¹²rAAV genome copies is appropriate. In certain preferred embodiments,10¹² rAAV genome copies is effective to target heart, liver, andpancreas tissues. In certain embodiments, the dosage of rAAV is 10¹⁰,10¹¹, 10¹², 10¹³, or 10¹⁴ genome copies per kg. In certain embodiments,the dosage of rAAV is 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ genomecopies per subject. In some cases, stable transgenic animals areproduced by multiple doses of a rAAV.

In some embodiments, rAAV compositions are formulated to reduceaggregation of AAV particles in the composition, particularly where highrAAV concentrations are present (e.g., ˜10¹³ GC/ml or more). Methods forreducing aggregation of rAAVs are well known in the art and, include,for example, addition of surfactants, pH adjustment, salt concentrationadjustment, etc. (See, e.g., Wright F R, et al., Molecular Therapy(2005) 12, 171-178, the contents of which are incorporated herein byreference.)

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound in eachtherapeutically-useful composition may be prepared is such a way that asuitable dosage will be obtained in any given unit dose of the compound.Factors such as solubility, bioavailability, biological half-life, routeof administration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Dispersions may also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. In many cases the form issterile and fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art. For example, one dosage may be dissolvedin 1 ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the host. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual host.

Sterile injectable solutions are prepared by incorporating the activerAAV in the required amount in the appropriate solvent with various ofthe other ingredients enumerated herein, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The rAAV compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts, include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Supplementary active ingredients can also be incorporated into thecompositions. The phrase “pharmaceutically-acceptable” refers tomolecular entities and compositions that do not produce an allergic orsimilar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles,microspheres, lipid particles, vesicles, and the like, may be used forthe introduction of the compositions of the present invention intosuitable host cells. In particular, the rAAV vector delivered transgenesmay be formulated for delivery either encapsulated in a lipid particle,a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids or therAAV constructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art. Recently, liposomes weredeveloped with improved serum stability and circulation half-times (U.S.Pat. No. 5,741,516). Further, various methods of liposome and liposomelike preparations as potential drug carriers have been described (; U.S.Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures. In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, drugs, radiotherapeutic agents, viruses, transcriptionfactors and allosteric effectors into a variety of cultured cell linesand animals. In addition, several successful clinical trails examiningthe effectiveness of liposome-mediated drug delivery have beencompleted.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 .ANG., containing an aqueous solution in the core.

Alternatively, nanocapsule formulations of the rAAV may be used.Nanocapsules can generally entrap substances in a stable andreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use.

In addition to the methods of delivery described above, the followingtechniques are also contemplated as alternative methods of deliveringthe rAAV compositions to a host. Sonophoresis (e.g., ultrasound) hasbeen used and described in U.S. Pat. No. 5,656,016 as a device forenhancing the rate and efficacy of drug permeation into and through thecirculatory system. Other drug delivery alternatives contemplated areintraosseous injection (U.S. Pat. No. 5,779,708), microchip devices(U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al.,1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) andfeedback-controlled delivery (U.S. Pat. No. 5,697,899).

Kits and Related Compositions

The agents described herein may, in some embodiments, be assembled intopharmaceutical or diagnostic or research kits to facilitate their use intherapeutic, diagnostic or research applications. A kit may include oneor more containers housing the components of the invention andinstructions for use. Specifically, such kits may include one or moreagents described herein, along with instructions describing the intendedapplication and the proper use of these agents. In certain embodimentsagents in a kit may be in a pharmaceutical formulation and dosagesuitable for a particular application and for a method of administrationof the agents. Kits for research purposes may contain the components inappropriate concentrations or quantities for running variousexperiments.

The kit may be designed to facilitate use of the methods describedherein by researchers and can take many forms. Each of the compositionsof the kit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the invention.

Instructions also can include any oral or electronic instructionsprovided in any manner such that a user will clearly recognize that theinstructions are to be associated with the kit, for example, audiovisual(e.g., videotape, DVD, etc.), Internet, and/or web-based communications,etc. The written instructions may be in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which instructions can alsoreflects approval by the agency of manufacture, use or sale for animaladministration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. In someembodiments, the kit comprises a container(s) housing agents(components) of a molecular sensing system. The agents may be in theform of a liquid, gel or solid (powder). The agents may be preparedsterilely, packaged in syringe and shipped refrigerated. Alternativelyit may be housed in a vial or other container for storage. A secondcontainer may have other agents prepared sterilely. Alternatively thekit may include the active agents premixed and shipped in a syringe,vial, tube, ampule or other container. The kit may have one or more orall of the components required to administer the agents to an animal,such as a syringe, topical application devices, or iv needle tubing andbag, particularly in the case of the kits for producing specific somaticanimal models.

EXAMPLES Example 1 rAAV-Mediated Delivery of microRNA Scavengers Leadsto Efficient and Stable Knock-Down of Cognate microRNA, Upregulation oftheir Natural Target Genes and Phenotypic Changes in Mice

The use of rAAV for the delivery of miRNA antagonists (miR-Antags) inadult mice was investigated. Different designs of vector backbone (ssversus sc), promoter (Pol II versus Pol III) and miRNA antagonist(Sponge, Zip, TuD, etc.) were evaluated for efficient somatic inhibitionof specific miRNAs. Different designs of miRNA antagonists (inhibitors)were also evaluated, e.g., bulged binding sites, multiple-tandem copysponges, etc. MiR-122, which has been reported to regulate cholesterolbiosynthesis in the liver, and an anti-oncogenic miRNA, Let-7, were usedas targets for inhibition. In order to select high function inhibitors,a chemiluminescent miRNA sensor was developed (See FIG. 1). Thechemiluminescent miRNA sensor contained a Polymerase II promoter drivingexpression of a reporter gene in a rAAV vector. The reporter gene had anintron just downstream of the promoter and a series of miRNA bindingsites (sponges) upstream of a poly-A tail. The polymerase II promoterand reporter gene were flanked by inverted terminal repeat sequences. AU6 promoter driving expression of the test miRNA inhibitor was presentin the intron of the reporter gene. Thus, the miRNA sensor comprisesdual miRNA regulators for sequential repression and derepression of thereporter gene and target validation.

The effectiveness of miR-122 tough decoy RNA designs were assessed. 293cells were infected with a miRNA sensor encoding β-galactosidase andexpressing miR122 tough decoy RNAs. A control miRNA sensor was alsotransfected that did not express miR122 decoys RNAs. The test andcontrol miRNA sensors each had 3 miR-122 binding sites. The 293 cellswere transfected with 0 ng, 50 ng, 100 ng, 200 ng, and 400 ng. LacZstaining was performed using standard techniques to evaluate reportergene expression. A dose dependent inhibition of reporter gene expressionwas observed in the control miRNA sensor. However, the test miRNA sensorwhich expressed miR122 inhibitor exhibited significant attenuation ofinhibition of the reporter gene expression at all doses. In contrast,cells infected with a miRNA sensor encoding β-galactosidase andexpressing miR122 sponge RNAs did not attenuate reporter gene expressioncompared with control miRNA sensors. Thus, the TuD miR122 design wassuperior to the sponge design. Similar experiments where performed inHuh7 cells which expressed steady-state levels of about 1.6×10⁴ miR-122molecules per cell. In Huh7 cells, the effect of miRNA binding sitenumber was evaluated. The test and control miRNA sensors each had either1 or 3 miR-122 binding sites. It was found that TuD miR-122 RNA (SEQ IDNO: 1) completely rescued the down-regulation associated with one copyof a miR-122 binding site behind the LacZ reporter gene in Huh-7 cells.Different combinations of promoters (Pol II and Pol III) and miRNAinhibitors were evaluated. Polymerase III driving expression of TuDmiR-122 inhibitors has superior results in both 293 and Huh-7 cells(FIG. 2A and B). A similar miRNA sensor having a firefly luciferase(Fluc) reporter gene was developed and tested in Huh-7 cells. Again, TuDmiR-122 RNA efficiently rescued the down-regulation of Fluc mediate bymiR-122 binding sites in Huh-7 cells.

Mice (adult B6) infected with rAAV (Serotype 9) harboring TuD miR-122inhibitor genes (for up to 7 weeks post infection) exhibited no adverseeffects on liver function, as assessed by liver enzymes levels.Effective induction of miR-122 target genes was observed in miceinfected with rAAV (Serotype 9) harboring TuD miR-122 inhibitor genesafter 1 month post infection, compared with control mice which wereinfected with rAAV9 harboring scrambled inhibitors. The target genesevaluated include Aldolase A, Cyclin G1, Tmed3 and Hfe2. MiR-122inhibitors delivered by rAAV9 had no effect on these target genes in themouse heart, the cells of which do not express miR122. A single IVinjection of rAAVmiR-122-Antag to C57BL/6 mice produced an 80% decreasein the level of mature miR-122 (FIG. 2C) and a 3-fold increase in themRNA levels of miR-122 target genes. Inhibition of miR-122 reduced totalserum cholesterol, HDL, and LDL by 50% in mice fed a regular diet. Thesequence and secondary structure of the TuD miR-122 inhibitor is shownin FIGS. 3A and 3B, respectively.

Similar experiments were performed to evaluate miRNA inhibitors ofLet-7. TuD Let-7 inhibitors were identified that can de-repressluciferase expression mediated by up to 7 copies of Let-7 spongesequences (Let-7 binding sites). A 2-fold increase in the expression ofDicer mRNA, a Let-7 target, was also observed. Similarly, TuD Let-7, butnot Let-7 sponges, induced Dicer protein levels in HeLa cells. Inductionof Dicer gene expression was also observed in mice liver and heartinfected with rAAV (Serotype 9) harboring TuD Let-7 inhibitor genes (forup to 7 weeks post infection) with no adverse effects on liver functionobserved. Administration of rAAV-Let-7-Antag increased by 2-fold themRNA levels of Dicer, the enzyme that produces miRNAs from pre-miRNAsand which is normally repressed by Let-7. The results of this studyindicate that rAAV-miR-Antags mediate efficient and stable somaticinhibition of miRNAs and will provide both an efficient tool to studymiRNA function as well as a potential therapeutic for dyslipidemia, inthe case of miR122, and other diseases caused by miRNA deregulation.

Example 2 rAAV-Mediated Therapeutic Silencing of miR-122 Leads to Rapidand Significant Reduction of LDL in DLDR^(-/-)/Apobec 1^(-/-) Mice

MicroRNA (miRNA) regulation was evaluated as an alternative to FH genetherapy. miRNAs play critical roles in regulating most cellularprocesses. The most abundant miRNA in the liver, miR-122 regulatescholesterol metabolism by an unknown mechanism(s) and does not directlytarget LDLR mRNA. Recombinant AAV9 was examined for efficient and stablemiR-122 antagonism in normal C57BL/6 mice by expressing an optimizedmiRNA-122 antagonist (Antag). A single intravenous injection ofrAAV9-miR-122Antag (SEQ ID NO: 1) produced an 80% decrease in the levelof mature miR-122 and 3-fold up-regulation of four miR-122 target genesas well as a 50% reduction in total serum cholesterol, HDL, and LDL inmale mice fed a regular diet (FIG. 4A, 4B, and 4C). This inhibition wasobserved for up to 14 weeks post infection with no significant impact onliver function as assessed by Alanine aminotransferase (ALT) andaspartate aminotransferase (AST) gene expression levels. ALT and AST areenzymes located in liver cells that leak out into the generalcirculation when liver cells are injured. To assess the therapeuticpotential of miR-122 inhibition, the same vector was administered toadult male and female LDLR-/-/Apobec 1A-/- mice, the most comparablemouse model of human FH with the normal chow diet (Powell-Braxton L, etal., Nature Medicine, Volume 4, Number 8, August 1998.) One week afterdosing, a 20% decrease in total serum cholesterol was observed in bothmales and females. Interestingly, the decreases in males wereexclusively in the HDL fraction, whereas the declines in females wereexclusively in LDL. By the second week, total cholesterol and LDL in thetreated females had declined about 30% but HDL levels remainedunchanged. (See, FIG. 5A, 5B, and 5C.) The reduction of totalcholesterol in males remained at 20%, reflecting a 50% increase in HDLand a 13% drop in LDL as compared to the mice in week 1. The observedsex-specific differences in miR-122 inhibition may reflect thepreviously reported lower efficiency of rAAV-mediated liver transductionin female mice, suggesting that doses may be optimized for rAAV-mediatedtherapeutic inhibition of miR-122 for the treatment of FH (Davidoff A M,et al., Blood. 2003;102:480-488). The results of this study indicatethat rAAV can achieve efficient and stable somatic miRNA inhibitionproviding basis for a therapy for dyslipidemia and other diseases causedby miRNA deregulation.

Example 3 AAV Vector-Mediated in Vivo miRNA Antagonism for TreatingHyperlipidemia

Genetic disruption of a miRNA gene represents a powerful strategy tostudy miRNA function, but many miRNA genes share the same seedsequence—the 6-8 nt miRNA region that defines its target repertoire—andtherefore one member of a miRNA family can compensate for loss ofanother. Creation of an animal model in which all members of a miRNAfamily are deleted is daunting. Moreover, humans and mice share morethan 276 miRNAs, requiring hundreds of conditional knockout strains toassess the function and contribution to disease of each conserved miRNAin adult mice. Chemically modified anti-miRNA oligonucleotides (AMO)complementary to mature miRNAs are widely available tools for miRNAinhibition in vitro and in vivo³⁻⁹. Effective AMOs typically employexpensive or proprietary chemical modifications such as 2′-O-methyl,2′-O-methoxyethyl, or 2′,4′-methylene (locked nucleic acid; LNA), andcurrent chemistries and formulations do not permit safe and effectivedelivery of AMOs to many tissues or organs. Additionally, miRNAinhibition with AMOs requires repeated administrations to suppressexpression of the cognate miRNA^(3,7-11).

As an alternative to AMOs, plasmid DNA vectors that express miRNA“sponges”—multiple, tandem miRNA binding sites designed to competitivelyinhibit miRNA function and expressed from an RNA polymerase IIpromoter—have been used to study miRNA function in cultured cells¹² andin vivo in flies¹³. Depletion of miR-223 in hematopoietic cells using asponge-expressing lentiviral vector to stably modify hematopoietic stemcells ex vivo, followed by bone marrow reconstitution in mice, produceda phenotype similar to that observed in a genetic miRNA knockout¹⁴.However, the risk of insertional mutagenesis and the requirement for exvivo manipulation may limit the use of the lentiviral vector-based miRNAinhibition for functional genomics studies and therapeutic applications.More recently, compact, RNA polymerase III-driven miRNA decoys have beenreported, including “Tough Decoy” (TuD) RNAs and miRZips, both of whichenable stable and permanent inhibition of miRNA in cultured cells and invivo¹⁶. Nevertheless, a method to stably and efficiently antagonizemiRNAs for studying miRNA-target interactions in adult mammals remainsto be developed.

The 4.7 kb single-stranded DNA parvovirus Adeno-associated virus¹⁷ (AAV)is a widespread, nonpathogenic resident in primates, includinghumans^(18,19). In the past decade, new recombinant AAV (rAAV) vectorshave been created from natural AAV serotypes, providing efficient genetransfer vehicles that target diverse tissues in mice and non-humanprimates²⁰⁻²³.

Here, the use of rAAV vectors in mice to inhibit miR-122, a miRNA highlyabundant in liver²⁴, and let-7, a miRNA with functions in cancer anddevelopment²⁵ is reported. Different promoters (RNA polymerase II versusRNA polymerase III) and designs of miRNA antagonists (sponge, TuD, andmiRZip) were evaluated in cultured cells, and RNA polymerase III-drivenTuD was identified as the most potent miRNA antagonist. rAAV9 vectorswere engineered expressing anti-miR-122 and anti-let-7 TuD RNAs and wereused to achieve efficient, sustained and target-specific miR-122 orlet-7 inhibition in vivo. Each miRNA inhibitor increased the expressionof the corresponding miRNA target genes in adult mice. High throughputsequencing of liver miRNAs from the treated mice confirmed that thetargeted miRNA, but no other miRNAs, were depleted. Moreover, miRNAdepletion in vivo was accompanied by the 3′ addition of non-templatednucleotides as well as 3′-to-5′ shortening of the miRNA, a degradationpathway previously observed in vivo in Drosophila melanogaster and invitro in transformed, cultured human cells³³. Importantly, sustainedphenotypic changes were observed in the serum cholesterol profiles ofboth wild-type C57BL/6 and low density lipoprotein (LDL)receptor-deficient mice treated with rAAV9-expressing the anti-miR-122,but not the anti-let-7, TuD RNA. The data provided herein suggest thatrAAV-expressing TuD RNAs could enable stable therapy forhypercholesterolemia and other disorders caused by miRNA expression.

Evaluation of Transcribed miRNA Antagonists in Cultured cells

To test different transcribed miRNA antagonists, a highly abundantmiRNA, miR-122, which regulates cholesterol biosynthesis in the liver,and an anti-oncogenic miRNA, let-7, were chosen as targets forinhibition. A series of miR-122 and let-7 antagonists were designedincluding miRNA sponges, TuD RNAs (FIG. 6) and miRZips^(12,15)(www.systembio.com/microrna-research/microma-knockdown/mirzip/) (Table2). miRNA sponges were expressed using the RNA polymerase II simianvacuolating virus (SV40) promoter, or the liver-specific, human thyroidhormone-binding globulin (TBG) promoter, or, alternatively, the RNApolymerase III U6 promoter; the U6 promoter was used to drive TuD andmiRZip expression (FIGS. 7 a and b).

TABLE 2 Oligonucleotide Sequence (5′ to 3′) anti-miR-122 TuDGGATCCGACGGCGCTAGGATCATCAACCAAACACCATTGATCTTCACACTCCACAAGTATTCTGGTCACAGAATACAACCAAACACCATTGATCTTCACACTCCACAAGATGATCCTAGCGCCGTCTTTTTTGAATTC (SEQ ID NO: 19) anti-let-7 TuDGGATCCGACGGCGCTAGGATCATCAACAACTATACAACCATCTTACTACCTCACAAGTATTCTGGTCACAGAATACAACAACTATACAACCATCTTACTACCTCACAAGATGATCCTAGCGCCGTCTTTTTTGAATTC (SEQ ID NO: 20) miR-122 miRZipGGATCCTGGTCAGTGACAATGTTTGCTTCCTGTCAGACAAACACCATTGTCACACTCCATTTTTAAGCTTGAAGACAA TAGC (SEQ ID NO: 21)anti-let-7 miRZip GGATCCTCTCGTAGTAGGTTGTATAGTTCTTCCTGTCAGAAACTATACAACCTACTACCTCATTTTTAAGCTTGAAGA CAATAGC (SEQ ID NO: 22)anti-miR-122 sponge TCTAGACAAACACCATACAACACTCCACAAACACCATACAACACTCCACAAACACCATACAACACTCCACAAACACCATACAACACTCCACAAACACCATACAACACTCCACAAACACCATACAACACTCCACAAACACCATACAACACTCC AGGGCCC (SEQ ID NO: 23)anti-let-7 sponge TCTAGAAACTATACAAAACCTACCTCAAACCACACAAAACCTACCTCAAACCATACAAAACCTACCTCAAACTATGCAAAACCTACCTCTAACTATACAAAACCTACCTCAAACTGTACAAAACCTACCTCAAACCATACAAAACCTACCTC AGCCCTAGA (SEQ ID NO: 24)Mutant anti-miR-122 TCTAGACAAACACCATACAACAAGAAACAAACACCATA spongeCAACAAGAAACAAACACCATACAACAAGAAACAAACACCATACAACAAGAAACAAACACCATACAACAAGAAACAAACACCATACAACAAGAAACAAACACCATACAACAA GAAAGGGCCC (SEQ ID NO: 25)Mutant anti-let-7 sponge TCTAGAAACTATACAAAACCTAAAGAAAACCACACAAAACCTAAAGAAAACCATACAAAACCTAAAGAAAACTATGCAAAACCTAAAGATAACTATACAAAACCTAAAGAAAACTGTACAAAACCTAAAGAAAACCATACAAAACCTAAAGA AGGGCCC (SEQ ID NO: 26)(miR-122)1 sense pCGAAACAAACACCATTGTCACACTCCATT (SEQ ID NO: 27)(miR-122)1 antisense pCGAATGGAGTGTGACAATGGTGTTTGTTT (SEQ ID NO: 28)(miR-122)3 sense pCGAAACAAACACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAA CAAACACCATTGTCACACTCCATT (SEQ ID NO: 29)(miR-122)3 anti-sense pCGAATGGAGTGTGACAATGGTGTTTGTTGGAGTGTGACAATGGTGTTTGTT GGAGTGTGACAATGGTGTTTGTTT (SEQ ID NO: 30)XbaI-ApaI linker F CTAGATTCCGAGATATCGGTAATGGGCC (SEQ ID NO: 31)XbaI-ApaI linker R GGCCCATTACCGATATCTCGGAATCTAG (SEQ ID NO: 32)pri-miR-122 F ATCGGGCCCGACTGCAGTTTCAGCGTTTG (SEQ ID NO: 33)pri-miR-122 R CGCGGGCCCGACTTTACATTACACACAAT (SEQ ID NO: 34) Nras FTGGACACAGCTGGACAAGAG (SEQ ID NO: 35) Nras RCTGTCCTTGTTGGCAAGTCA (SEQ ID NO: 36) Kras FCAAGAGCGCCTTGACGATACA (SEQ ID NO: 37) Kras RCCAAGAGACAGGTTTCTCCATC (SEQ ID NO: 38) Hras1 FCGTGAGATTCGGCAGCATAAA (SEQ ID NO: 39) Hras1 RGACAGCACACATTTGCAGCTC (SEQ ID NO: 40) Mm-Dicer FGCAGGCTTTTTACACACGCCT (SEQ ID NO: 41) Mm-Dicer RGGGTCTTCATAAAGGTGCTT (SEQ ID NO: 42) c-MYC FCAACGTCTTGGAACGTCAGA (SEQ ID NO: 43) c-MYC RTCGTCTGCTTGAATGGACAG (SEQ ID NO: 44) Hfe2 FGGGGACCTTGCTTTCCACTC (SEQ ID NO: 45) Hfe2 RGCCTCATAGTCACAGGGATCT (SEQ ID NO: 46) Tmed3 FAGCAGGGCGTGAAGTTCTC (SEQ ID NO: 47) Tmed3 RTTGTACGTGAAGCTGTCATACTG (SEQ ID NO: 48) Aldolase A FTGGGAAGAAGGAGAACCTGA (SEQ ID NO: 49) Aldolase A RAGTGTTGATGGAGCAGCCTT (SEQ ID NO: 50) CAT-1 FTACCAGTGGCCGTGTTTGTA (SEQ ID NO: 51) CAT-1 RGCTGTTGCCAAGCTTCTACC (SEQ ID NO: 52) Cyclin G1 FAATGGCCTCAGAATGACTGC (SEQ ID NO: 53) Cyclin G1 RAGTCGCTTTCACAGCCAAAT (SEQ ID NO: 54) Mm-Actin FATGCCAACACAGTGCTGTCTGG (SEQ ID NO: 55) Mm-Actin RTGCTTGCTGATCCACATCTGCT (SEQ ID NO: 56) miR-122 probeTGGAGTGTGACAATGGTGTTTG (SEQ ID NO: 57) Let-7 probeAACTATACAACCTACTACCTCA (SEQ ID NO: 58) miR-26a probeAGCCTATCCTGGATTACTTGAA (SEQ ID NO: 59) miR-22 ProbeACAGTTCTTCAACTGGCAGCTT (SEQ ID NO: 60) U6 probeCTCTGTATCGTTCCAATTTTAGTATA (SEQ ID NO: 61) IDT miRNA cloningAppCTGTAGGCACCATCAAT/ddC/ (SEQ ID NO: 62) linker-1 5′Illumina RNA Adapter GUUCAGAGUUCUACAGUCCGACGAUC (SEQ ID NO: 63)Small RNA RT primer ATTGATGGTGCCTACAG (SEQ ID NO: 64)Small RNA PCR Primer 1 CAAGCAGAAGACGGCATACGAATTGATGGTGCCTACAG(SEQ ID NO: 65) Small RNA PCR Primer2AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACA GTCCGA (SEQ ID NO: 66)

To evaluate the efficiency of each miRNA antagonist, the ability of theexpression constructs to de-repress a nuclear-targeted E. coliβ-galactosidase (nLacZ) reporter mRNA containing 1 or 3 copies of fullycomplementary miR-122-binding sites in the 3′ untranslated region (UTR)was tested. The nLacZ reporter plasmid was co-transfected with thevarious miR-122 inhibitor constructs or a control plasmid into HuH-7cells²⁷, a human hepatoma cell line expressing ˜16,000 miR-122 moleculesper cell²⁷. As expected, reporter expression was reduced ˜50% when onemiR-122-binding site was present in the nLacZ 3′ UTR and >80% when threesites were present (FIG. 7 c). Among the RNA polymerase II-drivenanti-miR-122 sponges, only the TBG promoter, a strong liver-specificpromoter, detectably increased expression of nLacZ bearing a singlemiR-122binding site, indicating that the sponge partially inhibitedmiR-122. However, nLacZ expression was not significantly increased bythis sponge when the reporter contained three miR-122-binding sites(FIG. 7 c), suggesting that the change in miR-122 activity orconcentration was too small to overcome the greater repression conferredby three miRNA target sites.

In contrast, both the one- and three-site reporters were de-repressed bythe RNA polymerase III-driven anti-miR-122 TuD RNA. For the one-sitereporter, the TuD restored nLacZ expression to that observed when nomiR-122 target sites were present in the reporter (FIG. 7 c). Thegreater efficacy of the TuD RNA might reflect the higher level oftranscription possible with RNA polymerase III compared to RNApolymerase II, greater miRNA inhibition by the TuD design, or both. Todistinguish among these possibilities, the ability of three differentU6-driven miR-122 antagonist constructs—sponge, TuD, and miRZip—tode-repress the nLacZ reporter containing three miR-122-binding sites wascompared. Again, only the TuD significantly (p-value≦0.001) derepressednLacZ repression by miR-122 in HuH-7 cells (FIG. 7 d). The anti-miR-122TuD expression construct was similarly effective in human embryonickidney (HEK) 293 cells. Because HEK 293 cells express little miR-122,pri-miR-122 was expressed from a plasmid, which was co-transfected alongwith the nLacZ reporter with or without three miR-122-binding sites andthe TuD-expressing plasmid. nLacZ expression was scored 48 h later. Theanti-miR-122 TuD, but not an anti-let-7 TuD or an anti-miR-122 oranti-let-7 sponge, significantly derepressed reporter expression in thepresence of the miR-122 expression plasmid (FIG. 7 e).

miRNAs that are extensively complementary to their targets directArgonaute2 protein to cleave the mRNA, whereas less extensivecomplementarity generally decreases mRNA stability. To test if the TuDRNA can also inhibit repression directed by a miRNA with imperfectcomplementarity to its target, a firefly luciferase (Fluc) reporter mRNAwas designed with seven copies of a bulged miR-122-binding site in its3′ UTR; Fluc with seven7 mutant sites served as a control. ThemiR-122-responsive Fluc reporter, anti-miR-122, anti-let-7 or controlTuD plasmid, and, as an internal control, a Renilla reniformisluciferase (Rluc) expression plasmid, were introduced into HuH-7 cellsby transfection. The anti-miR-122 TuD, but not the control or anti-let-7TuDs, fully de-repressed Fluc expression (FIG. 7 f). it is concludedthat TuD RNAs are potent and specific miRNA inhibitors.

Finally, the anti-let-7 TuD increased expression of both the Dicer mRNAand protein; dicer is an endogenous let-7 target^(28,29) (FIG. 8 a, 8 b,and FIG. 9). Together, the in vitro data suggest that the TuD RNAtranscribed from a U6 promoter was the most potent of the miRNAantagonists surveyed.

Real-Time Monitoring of Specific Endogenous miRNA Activities in LiveAnimals

To test the ability of TuD RNAs to inhibit miRNA function in vivo, aseries of rAAV vector genomes expressing a miRNA-responsive Gaussialuciferase (Gluc)³⁰ mRNA was constructed (FIG. 10 a). Gluc is a secretedprotein, enabling detection of the reporter in the blood or urine oflive animals. Seven bulged miR-122 or let-7 target sites were insertedinto the 3′ UTR of the Gluc mRNA to render it miRNA responsive. A U6promoter-driven expression cassette for either an anti-miR-122 or ananti-let-7 TuD RNA was inserted into the intron of the Gluctranscription unit. Reporter lacking either the seven miRNA-bindingsites or the TuD expression cassette or both served as controls. miR-122comprises 70% of total miRNAs in liver²⁷, posing a stringent test forthe ability of TuD RNAs to inhibit the function of even the mostabundant miRNA species. In vitro in HuH-7 cells, the anti-miR-122, butnot anti-let-7, TuD RNA derepressed the Gluc reporter bearing sevenmiR-122-binding sites (FIG. 10 b). Similarly, in HeLa cells anti-let-7TuD RNA derepressed the Gluc reporter bearing seven let-7binding sites(FIG. 10 c).

Both miR-122 and let-7 are present in liver²⁷, and let-7 is also foundin heart³¹. The rAAV genomes were packaged with the AAV9 capsid, whichpreferentially transduces liver and heart. To further improvetransduction, all rAAVs were prepared as self-complementary (sc)genomes³². The vectors were administered intravenously to adult maleC57B/6 mice and Gluc activity was monitored in blood. Initially, Glucactivity was comparable among the animals injected with vectorsexpressing the miR-122-regulated reporters, irrespective of the presenceof a TuD RNA expression cassette (days 3 and 7). By week 2, Glucactivity declined in the mice that received vectors lacking theantimiR-122 TuD, while Gluc activity increased in the mice treated withthe anti-miR-122 TuD expressing vector (FIG. 10 d). Similarly, Glucactivity was low in mice that received the let-7-regulated reporter andwas high in mice that received the same reporter containing theanti-let-7 TuD expression cassette. One notable difference between themiR-122- and let-7-regulated Gluc reporters was that the let-7-regulatedreporter was silenced at the earliest time point (day 3), whereas themiR-122-regulated reporter showed an initial lag in achieving silencing(FIG. 10 d.e). De-repression of Gluc expression by either anti-miR-122or anti-let-7 TuD RNA was sustained for the duration of the study, 18weeks (FIG. 10 d, e).

scAAV9-Delivered TuD RNAs Mediate Specific miRNA Depletion in MouseLiver

Four weeks after the administration of scAAV9 vectors, miRNA expressionwas analyzed in the liver using quantitative RT-PCR. An ˜80% reductionin miR-122 was observed in the mice that received the anti-miR-122 TuDexpressing vector, compared to vector expressing anti-let-7 TuD orcontrol vector lacking a TuD (FIG. 11 a). Northern blot analysisconfirmed the reduction of miR-122 in the mice that receivedanti-miR-122 TuD (FIG. 11 b and FIG. 12). let-7 was similarly reduced inthe mice treated with scAAV9 vectors expressing the anti-let-7 TuD (thelet-7 Northern probe employed cannot distinguish among the eight mouselet-7 isoforms). In contrast, no reduction was detected for miR-26a ormiR-22, two other abundant liver miRNAs (FIG. 11 b and FIG. 12).

High throughput sequencing of miRNAs from the treated livers furthersupports the view that scAAV9-delivered TuD RNAs effectively andspecifically trigger the destruction of complementary miRNAs. The TuDtargeting miR-122 (FIG. 7 b) reduced the abundance of full-length, 23 ntmiR-122 by 4.3-fold (FIG. 11 c), consistent with the qRT-PCR results(FIG. 11 a). The 21 and 22 nt miR-122 isoforms decreased less, whereas20 and 19 nt isoforms increased, suggesting that the TuD triggered3′-to-5′ exonucleolytic trimming of miR-122 (FIG. 11 c). Likeantagomir-directed destruction of miRNAs in human cell culture³³ theanti-miR-122 TuD promoted the addition of nontemplated nucleotides tothe 3′ end of miR-122 (FIG. 11 d). Prefix-matching reads—sequences thatinitially match the mouse genome but then end with non-templatednucleotides—doubled in the mouse expressing the anti-miR-122 TuD,compared to the control (FIG. 11 d). The 3′ non-templated nucleotidescomprised one or more adenosines. Even in the absence of the TuD, 30% ofmiR-122 was tailed with adenosine, suggesting that miR-122 undergoespost-transcriptional modification, perhaps as part of its naturalturnover.

Mouse liver expresses all eight let-7 isoforms (FIG. 13). These isoformsdiffer by 1-4 nucleotides outside their common seed sequence (FIG. 11e). Antilet-7 TuD strongly decreased the abundance of those full-lengthlet-7 isoforms that were fully complementary to the TuD sequence(let-7a, 12.1-fold) or contained only a single non-seed mismatch to theTuD (let-7c, 5.1-fold; let-7d, 5.0-fold; and let-7ƒ, 11.0-fold). Incontrast, the decrease was smaller for let-7b (1.6-fold) and let-7g(2.7-fold), which contain two 3′ mismatches to the TuD, let-7i(1.5-fold), which contains three 3′ mismatches to the TuD, and let-7e(3.6-fold), which contains a purine:purine mismatch to the TuD atposition 9, immediately flanking the seed sequence (FIG. 11 e, 11 f).Prefix-matching reads increased more for let-7a, c, d, and ƒ—the let-7isoforms that decreased the most in response to the anti-let-7TuD—whereas let-7 b, e, g and i, which decreased least showed noincrease in such trimmed-and-tailed species (FIG. 11 g). These findingsindicate that anti-let-7 TuD-directed miRNA decay requires nearlyperfect complementarity between TuD-RNA and the miRNA. For both theanti-miR-122 and the anit-let-7 TuDs, the overall abundance of othermiRNAs was unaltered (FIG. 14).

scAAV9-Delivered Anti-miRNA TuD RNAs Specifically Increase Expression ofEndogenous miRNA-Regulated mRNAs

When delivered using scAAV9, anti-miRNA TuD RNAs also de-repressmiR-122—and let-7-regulated endogenous mRNAs (FIG. 15). qRT-PCR was usedto analyze the expression of validated targets of miR-122 and let-7 inliver and heart four weeks after injection of the TuD-expressing scAAV9vectors. Mice injected with scAAV9 expressing the Gluc reporter but withno TuD RNA served as a control. For mice treated with the vectorexpressing anti-miR-122 TuD RNA, a 2.5 to 3.5-fold increase in AldolaseA (3.3±0.5; p-value≦0.04), Tmed3 (4.2±1.5; p-value≦0.01), Hƒe2 (3.3±1.0;p-value≦0.02), and Cyclin G1 (2.5±0.4; p-value≦0.001) mRNAs^(7,34) wasdetected in the liver, four genes previously shown to be regulated bymiR-122; expression of these four mRNAs was unaltered in the heart,which lacks miR-122 (FIG. 15). No statistically significant change inthe expression of the four miR-122-regulated mRNAs was found in eitherliver or heart from mice that received the vector expressing anti-let-7TuD RNA (FIG. 15).

The miRNA-producing enzyme Dicer²⁹ itself is repressed by let-7 familymiRNAs. qRT-PCR was used to measure Dicer mRNA abundance in mice thatreceived scAAV9 vector expressing either anti-miR-122 or anti-let-7 TuDRNA (FIG. 15). When let-7 was inhibited, Dicer mRNA was increased inboth liver (1.9±0.2; p-value ≦0.001) and heart (2.4±0.4; p-value≦0.003).The RAS family genes, HRAS, NRAS and KRAS, have been reported also to berepressed by the let-7 miRNA³⁵⁻³⁷. Increased expression of Nras wasobserved in both liver (1.3±0.1; p-value≦0.01) and heart (1.3±0.1;p-value≦0.02) and of Hras1 (1.3±0.1; p-value≦0.04) in heart in the micethat received scAAV9 expressing the anti-let-7, but not the anti-miR-122TuD RNA, relative to the control (FIG. 15).

Anti-miR-122 TuD RNA Reduces Cholesterol Levels

miR-122 is required for normal cholesterol biosynthesis; inhibition ofmiR-122 with AMOs decreases cholesterol metabolism in adultmice^(7,9,11) and non-human primates^(8,10). In wild-type mice, a singleintravenous injection of scAAV9 expressing anti-miR-122 RNAsignificantly reduced total serum cholesterol (45±5%; p-value≦0.001) andhigh-density lipoprotein (HDL, 42±5%; p-value≦0.001) levels beginningtwo weeks after injection, and this reduction was sustained for the 18week duration of the study. LDL levels were also reduced (88±102%;p-value≦0.05) by the third week and lasted for the duration of the study(FIG. 16 a). Total serum cholesterol, HDL, and LDL levels were unalteredin mice that received the anti-let-7 TuD. The body weight and liverfunction of the mice were normal throughout the study: no weight loss(FIG. 17) or statistically significant increase in serum alanineaminotransferase (ALT) or aspartate aminotransferase (AST) levels wasdetected (FIG. 16 b).

High cholesterol is a major risk factor for cardiovascular disease, themost common cause of morbidity and mortality in the United States.Mutations in the LDL receptor (LDLR) gene cause the common inheriteddyslipidemia, familial hypercholesterolemia³⁸. rAAV-mediated replacementof the LDL receptor represents a promising approach for the treatment ofthis genetic disorder, but may be limited by host immunity against thetherapeutic gene product^(39,40) . Sustained miR-122 inhibition couldprovide an alternative therapy for familial hypercholesterolemia.scAAV9-delivered anti-miR-122 TuD RNA was evaluated as potentialtreatment for familial hypercholesterolemia using a mouse model of thehuman disease: LDLR^(-/-), Apobec 1A^(-/-) double mutant mice fed anormal chow diet⁴¹. One month after a single intravenous dose of scAAV9expressing anti-miR-122 TuD RNA, total serum cholesterol was reduced by34±3% (p-value≦0.0006), serum HDL decreased 18±2% (p-value≦0.02), andserum LDL, which is the therapeutic target for familialhypercholesterolemia in female mice, decreased 53±6% (p-value≦0.006),compared to mice that received the scrambled TuD control (FIG. 16 c). Inmale mice, a 21±1% (p-value≦0.05) reduction in total cholesterol, a26±2% (p-value≦0.004) reduction in HDL, and a 20±1% (p-value≦0.02)reduction in LDL was measured (FIG. 16 c). The observed sex specificdifferences in lowering cholesterol in the LDLR^(-/-), Apobec 1A^(-/-)mice warrant further investigation.

DISCUSSION

The large number of mammalian miRNAs makes identifying their biologicalfunctions a daunting challenge. Inhibitors of miRNA function promise toaccelerate the understanding of miRNA biology, especially in adultmammals. Strategies to inhibit miRNAs include complementary chemicallymodified oligonucleotides and transcribed miRNA-binding competitor RNAs.While effective miRNA inhibitors, chemically modified oligonucleotidesare currently expensive, some modifications are not commerciallyavailable, and require repeated dosing that risks long-term toxicity.Moreover, many tissues are not currently accessible to delivery ofoligonucleotides.

Transcribed miRNA-binding RNAs provide an alternative tooligonucleotides. The small size of their transcripts makes them readilyincorporated into a variety of gene transfer vectors. PrimateAAV-derived vectors represent attractive tools for this applicationbecause of their unique tissue tropism, high efficiency of transduction,stability of in vivo gene transfer, and low toxicity^(22,42).

Recently, several designs of miRNA antagonists—sponges, TuD RNAs, andmiRZips—have been developed and tested in lentiviral vectors invitro^(12,15) and in genetic knockout animal models in vivo^(13,14,16).These miRNA antagonists were compared in vitro, and the most effectivedesign, the TuD RNAs, was used in vivo to inhibit miR-122 and let-7 byincorporating TuD expression cassettes into scAVV9. The data providedherein demonstrate that a single administration of rAAV9 expressing aTuD RNA provides a stable and efficient reduction in the level of thetargeted miRNA (FIG. 11), leading to an increase in expression of itsendogenous target mRNAs (FIGS. 8 and 15), and a corresponding phenotypicchange in metabolism (FIG. 16). The high throughput sequencing dataprovided herein suggest that, in mice, TuD RNAs inhibit their miRNAtargets via the same target-RNA directed tailing and trimming pathway asrecently described in flies for engineered³³ and endogenous mRNAs⁴³ andfor synthetic oligonucleotide “antagomirs” in cultured human HeLacells³³ (FIG. 11). The data presented here, which are the firstobservations of target RNA-directed miRNA tailing, trimming, anddestruction in a living mammal, suggest that this pathway may be widelyconserved among animals.

To date, methods to monitor miRNA function in live adult mammals havenot been described. The in vivo Gluc sensor system described hereprovides a simple means to detect changes in specific miRNA function,such as those caused by miRNA inhibitors (FIG. 10). This system allowsone to assess the activity of a specific miRNA in vitro in a cell lineor in vivo in a tissue or organ, providing a quantitative measure of theeffectiveness of a miRNA antagonist in live animals across time.

Retrospective profiling has linked aberrant miRNA expression to avariety of diseases, suggesting that miRNAs may provide new targets fortherapy⁴⁴⁻⁴⁸. Indeed, miR-122 inhibition by AMOs⁷⁻¹¹ or scAAV-deliveredTuD RNA (FIG. 16) lowers both HDL and LDL. However, the current viewthat HDL protects against heart attack⁴⁹ argues that therapy fordyslipidemia should lower LDL but raise HDL levels. Recently, miR-33 wasidentified as a repressor of HDL biogenesis; miR-33 inhibition raisesserum HDL level¹⁶. Perhaps simultaneous inhibition of miR-122 and miR-33by a pair of TuD RNAs expressed from a single scAAV vector may achieve amore balanced and healthy cholesterol profile and provide long-lastingtherapy for familial hypercholesterolemia.

Low miR-122 levels have been associated with hepatocellular carcinoma inrodents and humans⁵⁰⁻⁵², although no direct causal link has beenestablished^(51,52). Because AAV vector expression is stable for yearsin rodent and primate models, animals treated with scAAV9 expressinganti-miR-122 should enable testing the safety of prolonged miR-122inhibition in general and the increased risk of developinghepatocellular carcinoma in particular.

Materials and Methods Construction of miRNA Antagonist and SensorPlasmids

siFluc fragment in pRNA-U6.1/Neo-siFluc (GenScript, Piscataway, N.J.)was replaced with TuD miR-122, TuD let-7, miR-122Zip, let-7Zip, miR-122sponge and let-7 sponge that were designed as previouslydescribed^(12,15)(http://www.systembio.com/microrna-research/microma-knockdown/mirzip/)to generate U6-driven expression cassettes for expression of differentmiRNA antagonists. The design of let-7 antagonist was based on theconsensus sequence of all let-7 family members. The XbaI-ApaI linker wasgenerated by annealing oligonucleotide pairs, XbaI-ApaI linker F andXbaI-ApaI linker R (Table 2) followed by cloning into the ApaI siteafter Fluc gene in pGL3-control plasmid. The chemically synthesizedmiR-122 or let-7 sponge sequence flanked with XbaI and ApaI sites wasdigested and cloned into pGL3-XbaI-ApaI linker plasmid to create SV40promoter-driven sponge expression cassettes. Then, the fragmentcontaining Fluc gene and miR-122 or let-7 sponge was isolated by NcoIand ApaI double digestions from pGL3 miR-122 sponge or pGL3 let-7 spongeand cloned into the KpnI site of pAAVCBPI vector plasmid or between PstIand MluI sites of pAAVTBGPI vector plasmid to generate CB promoter andTBG promoter driven sponge expression vectors, respectively.

One or three copies of perfectly complementary miRNA target sites weredesigned based on the annotated miRNA sequences in miRBase⁵³ andinserted into the BstBI restriction site in the 3′ UTR of the nLacZexpression cassette of the ubiquitously-expressed pAAVCBnuclear-targeted β-galactosidase (nLacZ) plasmid using syntheticoligonucleotides (Table 2). To express miR-122, pri-miR-122 fragment wasamplified by PCR from mouse genomic DNA with specific oligonucleotides(Table 2), cloned into the XbaI restriction site right after a fireflyluciferase cDNA in the pAAVCB Fluc plasmid. The identity of pri-miR-122was verified by sequencing. scAAV9 vectors used in this study weregenerated, purified, and titered as previously described¹⁸.

To create AAV vectors, seven copies of bulged target sites for miR-122or let-7 were synthesized and cloned into BclI site after the Glucreporter gene in the pscAAVCBPI Gluc plasmid. The EcoRI and HindIIIfragment harboring U6-TuDmiR-122 or U6-TuD let-7 expression cassette wasisolated from pRNA-U6.1/Neo-TuDmiR-122 or pRNA-U6.1/Neo-TuD let-7plasmid and cloned into PpuMI site in the intron region of pscAAVCBPIGluc with or without bulged target sites for miR-122 or let-7.

Cell Culture

HEK 293, HuH-7 and HeLa cells were cultured in Dulbecco's Modified EagleMedium supplemented with 10% FBS and 100 mg/L of penicillin-streptomycin(HyClone, South Logan, Utah). Cells were maintained in a humidifiedincubator at 37° C. and 5% CO2. Plasmids were transiently transfectedinto cells using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) inaccordance with the manufacturer's instructions.

Luciferase Reporter Assay

Cells were lysed with passive lysis buffer (Dual-Glo Luciferase AssaySystem, Promega, Madison, Wis.) and 10 μl of lysis was used for theassay. Firefly and Renilla luciferase activities were assessed using theDual-Glo Luciferase Assay System (Promega, Madison, Wisc.) in accordancewith the manufacturer's instructions. The Gaussia luciferase (Gluc)assay was performed following the procedure described previously³⁰.Briefly, 10 μl each of culture media from the indicated transfectionswas used for the in vitro Gluc assay. To monitor Gluc expression invivo, the study animals were bled from a superficial cut on facial veinmade by a 5.5 mm animal lancet (MEDIpoint, Mineola, N.J.) at differenttime points after AAV9 vector treatment. Five μl each of blood sampleswas used for the Gluc assay.

Mice

C57BL/6 mice (Charles River Laboratories) and LDLR^(-/-)/Apobec 1A^(-/-)mice (Dr. James Wilson, University of Pennsylvania) were maintained andused for the study according to the guidelines of the InstitutionalAnimal Care and Use Committee of the University of Massachusetts MedicalSchool. Four-to-six weeks old wild type C57BL/6 male mice were treatedwith AAV vectors at 1×10¹² genome copies/mouse or 5×10¹³ genomecopies/kg by tail vein injection. To evaluate therapeutic potential ofscAAV9TuDmiR122, 4 to 6 weeks old LDLR^(-/-)/Apobec 1A^(-/-) mice weretreated with TuD-miR-122 or Scrambled vector at a dose of 3×10¹¹ genomecopies/mouse or 1.5×10¹³ genome copies/kg by tail vein injection. Tomonitor lipid profiles of the study animals, the serum samples werecollected at different times after AAV9 vector injection and analyzedfor total cholesterol, HDL and LDL on a COBAS C 111 analyzer (RocheDiagnostics, Lewes, UK). For RNA analyses, the animals were necropsiedat 4 weeks after the treatment; liver and heart tissues were harvestedfor RNA preparation.

qRT-PCR Analysis

RNA was extracted using Trizol (Invitrogen Carlsbad, Calif.), accordingto the manufacturer's instructions. Total RNA (0.5-1 μg) was primed withrandom hexamers and reverse-transcribed with MultiScribe ReverseTranscriptase (Applied Biosystems, Foster City, Calif.). QuantitativePCR reactions were performed in triplicate with 0.3 μM gene specificprimer pairs (Table 2) using the GoTaq qPCR master mix (Promega,Madison, Wis.) in a StepOne Plus Real-time PCR device (AppliedBiosystems, Foster City, Calif.). The expression of mature miR-122 andU6 was assayed using the TaqMan microRNA Assay (Applied Biosystems,Foster City, Calif.).

Northern Blot Analysis

To detect miR-122, miR-26a, miR-22 and let-7 in total liver RNA, 10 μgof total RNA was resolved by denaturing 15% polyacrylamide gels,transferred to Hybond N+ membrane (Amersham BioSciences, Pittsburgh,Pa.), and cross-linked with 254 nm light (Stratagene, La Jolla, Calif.).Synthetic DNA oligonucleotides (Table 2), 5′ end-labeled with γ-³² P ATPusing T4 polynucleotide kinase (NEB, Beverly, Mass.), were used asprobes for miR-122, miR-26a, miR-22 and let-7 and U6 (Table 2) andhybridized in Church buffer (0.5 M NaHPO4, pH 7.2, 1 mM EDTA, 7% [w/v]SDS) at 37° C. Membranes were washed using 1×SSC, 0.1% (w/v) SDS buffer,and then visualized using a FLA-5100 Imager (FUJIFILM, Tokyo, Japan).

Small RNA Sequencing

Small RNA libraries were constructed and sequenced as described³³.Briefly, 50 μg total RNA was isolated with the mirVana kit (AmbionFoster City, Calif.), 19-29 nt small RNAs were separated and isolatedthrough gel electrophoresis using 15% polyacrylamide/urea gel (SequaGel,National Diagnostics, Atlanta, Ga.). IDT miRNA cloning linker-1 wasligated to the 3′ of small RNAs using truncated T4 RNA ligase 2 (NEB,Beverly, Mass.) and gel purified; a 5′ RNA adapter was ligated to the 3′ligated RNA with T4 RNA ligase (NEB, Beverly, Mass.). The ligationproduct was used as template for reverse transcription with Small RNA RTprimer. The cDNA was amplified with small RNA PCR primer 1 and RNA PCRprimer 2. The PCR product was gel-purified and submitted for highthroughput sequencing. For sequencing statistics see Tables 3 and 4.Small RNA analyses were as previously described³³. Sequence data areavailable through the NCBI Short Read Archive(www.ncbi.nlm.nih.gov/sites/sra) as GSE25971.

TABLE 3 Sequencing statistics: Analysis of 5′ prefix-matching reads. Todetect small RNAs bearing 3′ terminal, non-templated nucleotides, readsmatching the reference genome for only part of their entire length wereidentified. Reads Reads Small RNA Pre perfectly matching reads miRNATotal matching annotated (excluding matching Sample reads genome ncRNAsncRNAs) reads Control 3,239,264 2,335,379 8,894 2,326,485 2,174,544anti-miR- 3,386,944 2,189,155 16,366  2,172,789 1,917,478 122 TuDanti-let-7 1,893,012 1,232,744 4,021 1,228,723 1,163,466 TuD

TABLE 4 Prefixes Prefixes Prefixes excluding matching Prefixes Pre miRNAmatching internal annotated (excluding matching Sample Total readsgenome mm ncRNAs ncRNAs) prefixes Control 3,239,264 903,885 800,5191,920 798,599 568,087 Anti-miR-122 TuD 3,386,944 1,197,478   1,086,409  2,855 1,083,554   775,181 Anti-let-7 TuD 1,893,012 660,268 574,454 1,008573,446 344,092

Western Blot Analysis

Proteins were extracted with RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mMNaCl, 1% NP40 [v/v], 1% sodium deoxycholate [w/v], 0.1% SDS [w/v])containing a protease inhibitor mixture (Boston BP). Proteinconcentration was determined using the Bradford method. Protein samples,50 μg each, were loaded onto 10% polyacrylamide gels, electrophoresed,and transferred to nitrocellulose membrane (Amersham BioSciences,).Immunoblotting was performed using the LI-COR infrared imaging system.Briefly, membranes were blocked with blocking buffer (LI-COR) at roomtemperature for 2 h, followed by incubation with either anti-GAPDH(Millipore), anti-Dicer (Santa Cruz) for 2 h at room temperature. Afterthree washes with PBS plus 0.1% Tween-20 (v/v), membranes were incubatedfor 1 h at room temperature using secondary antibodies conjugated toLI-COR IRDye. Signals were detected using the Odyssey Imager (LICOR).

Statistical Analysis

All results are given as mean±standard deviation and compared betweengroups using the two-tailed Student's t-test, except in FIG. 16 c, wherethe p-value was calculated using the Mann-Whitney test.

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The entire contents of references 1-53 listed above, and of all otherreferences, publications, or database entries identified herein areincorporated into this application by reference as if each individualreference, publication, or database entry was incorporated herein byreference individually. In case of a conflict, the instant disclosureshall control.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thisdescription or illustrated in the drawings. The invention is capable ofother embodiments and of being practiced or of being carried out invarious ways. Also, the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having,” “containing,”“involving,” and variations thereof herein, is meant to encompass theitems listed thereafter and equivalents thereof as well as additionalitems.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

1. A method for treating a high cholesterol-related disorder in asubject, the method comprising: administering an effective amount of arecombinant Adeno-Associated Virus (rAAV) to the subject, wherein therAAV comprises at least one transgene that expresses a miRNA inhibitorthat inhibits the expression of miR-122 in the subject.
 2. The method ofclaim 1, wherein the miRNA inhibitor comprises an miR-122 binding site.3. The method of claim 1, wherein the miR-122 binding site is flanked bytwo stem sequences.
 4. The method of claim 1, wherein the miR-122binding site comprises a non-binding, central portion that is notcomplementary with miR-122, flanked by two portions that arecomplementary with miR-122.
 5. The method of claim 1, wherein the miRNAinhibitor comprises a first miR-122 binding site and a second miR-122binding site, wherein a first stem sequence flanks the first miR-122binding site at its 5′-end, a second stem sequence flanks the firstmiR-122 binding site at its 3′-end and the second miR-122 binding siteat its 5′-end, and a third stem sequence flanks the second miR-122binding site at its 3′-end. 6-17. (canceled)
 18. The method of claim 1,wherein the miRNA inhibitor comprises two or more miR-122 binding sites.19. The method of claim 1, wherein the miRNA inhibitor comprises orconsists of a sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 5, SEQID NO: 21, or SEQ ID NO:
 23. 20. The method of claim 1, wherein the rAAVhas a capsid of the AAV9 serotype, which has a sequence as set forth inSEQ ID NO:
 3. 21. The method of claim 1, wherein the rAAV has a capsidthat is a variant of the capsid of the AAV9 serotype.
 22. The method ofclaim 21, wherein the rAAV has a capsid of the AAV9 serotype variant,Csp-3, which has a sequence as set forth in SEQ ID NO:
 4. 23. The methodof claim 1, wherein the rAAV targets liver tissue.
 24. The method ofclaim 1, wherein the rAAV transduces hepatocytes.
 25. (canceled)
 26. Themethod of claim 1, wherein administering is performed intravenously.27-35. (canceled)
 36. A nucleic acid vector comprising: (a) a firstpromoter operably linked with a transgene that comprises: (i.) a proteincoding region, and (ii.) at least one binding site of a test miRNA; and(b) a second promoter operably linked with a miRNA inhibitor codingregion, wherein the miRNA inhibitor specifically binds to the testmiRNA.
 37. The nucleic acid vector of claim 36, wherein the firstpromoter is a RNA Polymerase II promoter.
 38. The nucleic acid vector ofclaim 36, wherein the second promoter is a RNA Polymerase III promoter.39. The nucleic acid vector of claim 36 further comprising a firstuntranslated region between the first promoter and at least a portion ofthe protein coding region, wherein the second promoter and the miRNAinhibitor coding region are positioned within the first untranslatedregion. 40-41. (canceled)
 42. The nucleic acid vector of claim 36,wherein the transgene further comprises a second untranslated region,wherein the at least one binding site of the test miRNA is in the seconduntranslated region.
 43. (canceled)
 44. The nucleic acid vector of claim36 further comprising a pair of inverted terminal repeats that flank thefirst promoter and the transgene.
 45. (canceled)
 46. The nucleic acidvector of claim 36, wherein the protein coding region encodes a reporterprotein selected from: a fluorescent protein, luciferase,β-galactosidase, secreted alkaline phosphatase, β-glucuronidase,chloramphenicol acetyltransferase (CAT), and β-lactamase.
 47. A methodfor assessing the effectiveness of a miRNA inhibitor, the methodcomprising: (a) transfecting a cell with a nucleic acid vector of claim36, wherein the miRNA inhibitor coding region encodes the miRNAinhibitor; and (b) determining the level of expression of the proteinencoded by the protein coding region in the cell, wherein the level ofexpression of the protein is indicative of the effectiveness of themiRNA inhibitor. 48-49. (canceled)
 50. The method of claim 36, whereinthe test miRNA is a mammalian miRNA.
 51. The method of claim 36, whereinthe test miRNA is a human miRNA.
 52. The method of claim 36, wherein themiRNA inhibitor is a miRNA sponge, an antisense oligonucleotide, or atough decoy RNA.
 53. A method for assessing the effectiveness of a miRNAinhibitor, the method comprising: (a) transfecting a first cell with anucleic acid vector of, claim 36 wherein the miRNA inhibitor codingregion encodes the miRNA inhibitor; (b) transfecting a second cell withthe nucleic acid vector, wherein levels of the test miRNA are lower inthe second cell compared with the first cell; and (c) comparing thelevel of expression of the protein encoded by the protein coding regionin the first cell with the level of expression of the protein encoded bythe protein coding region in the second cell, wherein the results of thecomparison in (c) are indicative of the effectiveness of the miRNAinhibitor.
 54. A method for assessing the effectiveness of a miRNAinhibitor, the method comprising: (a) transfecting a cell with a nucleicacid vector of claim 36, wherein the miRNA inhibitor coding regionencodes the miRNA inhibitor; (b) determining a first level of expressionof the protein encoded by the protein coding region in the cell; (c)contacting the cell with the test miRNA; (d) determining a second levelof expression of the protein encoded by the protein coding region in thecell; and (e) comparing the first level of expression of the proteinwith the second level of expression, wherein the results of thecomparison in (e) are indicative of the effectiveness of the miRNAinhibitor.
 55. A kit for assessing the function of a miRNA inhibitor,the kit comprising: a container housing a nucleic acid vector of claim36.
 56. A kit for assessing the function of a miRNA inhibitor, the kitcomprising: a container housing a component of a molecular sensingsystem.
 57. A miRNA inhibitor comprising: a first miRNA binding site anda second miRNA binding site, wherein a first stem sequence flanks thefirst miRNA binding site at its 5′-end, a second stem sequence flanksthe first miRNA binding site at its 3′-end and the second miRNA bindingsite at its 5′-end, and a third stem sequence flanks the second miRNAbinding site at its 3′-end, wherein at least one of the miRNA bindingsites comprises a nucleotide sequence complementary to a consensussequence of the plurality of target miRNAs.
 58. The miRNA inhibitor ofclaim 57, wherein each of the two miRNA inhibitor binding sitescomprises a non-binding, central portion that is not complementary withany of the plurality of miRNAs targeted by the miRNA inhibitor. 59-69.(canceled)
 70. The miRNA inhibitor of claim 57, wherein the miRNAinhibitor comprises two or more miRNA binding sites.
 71. The miRNAinhibitor of claim 57, wherein the miRNA inhibitor is a TuD.
 72. ThemiRNA inhibitor of claim 57, wherein the miRNA inhibitor targets aplurality of let-7 family members.
 73. The miRNA inhibitor of claim 71,wherein the miRNA inhibitor comprises a sequence of at least 8contiguous nucleotides of SEQ ID NO:
 18. 74. (canceled)
 75. The miRNAinhibitor of claim 57, wherein the miRNA inhibitor comprises or consistsof the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO:
 20. 76. Amethod, comprising obtaining a consensus sequence from a plurality ofmiRNAs, and generating a miRNA inhibitor targeting a plurality ofmiRNAs, wherein the miRNA inhibitor comprises a first miRNA binding siteand a second miRNA binding site, wherein a first stem sequence flanksthe first miRNA binding site at its 5′-end, a second stem sequenceflanks the first miRNA binding site at its 3′-end and the second miRNAbinding site at its 5′-end, and a third stem sequence flanks the secondmiRNA binding site at its 3′-end, wherein the miRNA inhibitor comprisesa nucleotide sequence complementary to the consensus sequence of theplurality of target miRNAs.
 77. The method of claim 76, wherein theconsensus sequence is between 5 and 40 nucleotides long.
 78. The methodof claim 76, wherein the miRNA is a TuD.
 79. A miRNA inhibitorcomprising a nucleotide sequence of SEQ ID NO: 22, or SEQ ID NO:
 24. 80.A method comprising contacting a cell with an miRNA inhibitor of claim57.